Electrodes linked via conductive oligomers to nucleic acids

ABSTRACT

The invention relates to nucleic acids covalently coupled to electrodes via conductive oligomers. More particularly, the invention is directed to the site-selective modification of nucleic acids with electron transfer moieties and electrodes to produce a new class of biomaterials, and to methods of making and using them.

FIELD OF THE INVENTION

[0001] The invention relates to nucleic acids covalently coupled toelectrodes via conductive oligomers. More particularly, the invention isdirected to the site-selective modification of nucleic acids withelectron transfer moieties and electrodes to produce a new class ofbiomaterials, and to methods of making and using them.

BACKGROUND OF THE INVENTION

[0002] The detection of specific nucleic acids is an important tool fordiagnostic medicine and molecular biology research. Gene probe assayscurrently play roles in identifying infectious organisms such asbacteria and viruses, in probing the expression of normal genes andidentifying mutant genes such as oncogenes, in typing tissue forcompatibility preceding tissue transplantation, in matching tissue orblood samples for forensic medicine, and for exploring homology amonggenes from different species.

[0003] Ideally, a gene probe assay should be sensitive, specific andeasily automatable (for a review, see Nickerson, Current Opinion inBiotechnology 4:48-51 (1993)). The requirement for sensitivity (i.e. lowdetection limits) has been greatly alleviated by the development of thepolymerase chain reaction (PCR) and other amplification technologieswhich allow researchers to amplify exponentially a specific nucleic acidsequence before analysis (for a review, see Abramson et al., CurrentOpinion in Biotechnology, 4:41-47 (1993)).

[0004] Specificity, in contrast, remains a problem in many currentlyavailable gene probe assays. The extent of molecular complementaritybetween probe and target defines the specificity of the interaction.Variations in the concentrations of probes, of targets and of salts inthe hybridization medium, in the reaction temperature, and in the lengthof the probe may alter or influence the specificity of the probe/targetinteraction.

[0005] It may be possible under some limited circumstances todistinguish targets with perfect complementarity from targets withmismatches, although this is generally very difficult using traditionaltechnology, since small variations in the reaction conditions will alterthe hybridization. New experimental techniques for mismatch detectionwith standard probes include DNA ligation assays where single pointmismatches prevent ligation and probe digestion assays in whichmismatches create sites for probe cleavage.

[0006] Finally, the automation of gene probe assays remains an area inwhich current technologies are lacking. Such assays generally rely onthe hybridization of a labelled probe to a target sequence followed bythe separation of the unhybridized free probe. This separation isgenerally achieved by gel electrophoresis or solid phase capture andwashing of the target DNA, and is generally quite difficult to automateeasily.

[0007] The time consuming nature of these separation steps has led totwo distinct avenues of development. One involves the development ofhigh-speed, high-throughput automatable electrophoretic and otherseparation techniques. The other involves the development ofnon-separation homogeneous gene probe assays.

[0008] PCT application WO 95/15971 describes novel compositionscomprising nucleic acids containing electron transfer moieties,including electrodes, which allow for novel detection methods of nucleicacid hybridization.

SUMMARY OF THE INVENTION

[0009] Accordingly, it is an object of the invention to provide forimproved compositions of nucleic acids covalently attached to electrodesand at least one other electron transfer moiety.

[0010] In one aspect, the present invention provides compositionscomprising (1) an electrode; (2) at least one nucleoside; and (3) aconductive oligomer covalently attached to both said electrode and saidnucleoside. The conductive oligomer has the formula:

[0011] wherein

[0012] Y is an aromatic group;

[0013] n is an integer from 1 to 50;

[0014] g is either 1 or zero;

[0015] e is an integer from zero to 10; and

[0016] m is zero or 1;

[0017] wherein when g is 1, B-D is a conjugated bond; and

[0018] wherein when g is zero, e is 1 and D is preferably carbonyl, or aheteroatom moiety, wherein the heteroatom is selected from oxygen,sulfur, nitrogen or phosphorus.

[0019] In an additional aspect, the conductive oligomer has the formula:

[0020] wherein

[0021] n is an integer from 1 to 50;

[0022] m is 0 or 1;

[0023] C is carbon;

[0024] J is carbonyl or a heteroatom moeity, wherein the heteroatom isselected from the group consisting of nitrogen, silicon, phosphorus,sulfur; and

[0025] G is a bond selected from alkane, alkene or acetylene.

[0026] In one aspect, the present invention provides compositionscomprising (1) a first electron transfer moiety comprising an electrode;(2) a nucleic acid with a covalently second electron transfer moiety;and (3) a conductive oligomer covalently attached to both the electrodeand the nucleoside. The conductive oligomer may have the structuresdepicted above.

[0027] In an additional aspect, the invention provides methods ofdetecting a target sequence in a nucleic acid sample. The methodcomprises hybridizing a probe nucleic acid to the target sequence, ifpresent, to form a hybridization complex. The probe nucleic acidcomprises a conductive oligomer covalently attached to (1) a firstelectron transfer moiety comprising an electrode and (2) a singlestranded nucleic acid capable of hybridizing to the target sequence andcomprising a covalently attached second electron transfer moiety. Themethod further comprises the step of detecting electron transfer betweenthe electrode and the second electron transfer moiety, if present, as anindicator of the present or absence of said target sequence. Theconductive oligomer has the formula:

[0028] In a further aspect, the invention provides methods of detectinga target sequence in a nucleic acid wherein the target sequencecomprises a first target domain and a second target domain. The methodcomprises hybridizing a first probe nucleic acid to the first targetdomain, if present, to form a hybridization complex. The first probenucleic acid comprises a conductive oligomer covalently attached to (1)a first electron transfer moiety comprising an electrode and (2) asingle stranded nucleic acid capable of hybridizing to the targetsequence. Then, a second single stranded nucleic acid comprising acovalently attached electron transfer moiety to the second targetdomain, and electron transfer is detected between said electrode andsaid second electron transfer moiety, if present, as an indicator of thepresent or absence of said target sequence. The conductive oligomer canhave the structures outlined herein.

[0029] In an additional aspect, the present invention provides methodsfor attaching a conductive oligomer to a gold electrode comprisingadding an ethyl pyridine protecting group to a sulfur atom attached to afirst subunit of the conductive oligomer. The method may furthercomprise adding additional subunits to form the conductive oligomer. Themethod may additionally comprise adding at least first nucleoside to theconductive oligomer. The method may further comprise adding additionalnucleosides to said first nucleoside to form a nucleic acid. The methodmay additionally comprise attaching the conductive oligomer to the goldelectrode.

[0030] The invention also provides methods of making the compositions ofthe invention comprising providing a conductive oligomer covalentlyattached to a nucleoside; and attaching said conductive oligomer to saidelectrode. Alternatively, the compositions may be made by attaching aconductive oligomer to an electrode; and attaching at least onenucleotide to the conductive oligomer.

[0031] The invention additionally provides compositions comprising aconductive oligomer covalently attached to a nucleoside, wherein saidconductive oligomer has the formula:

[0032] The invention further provides compositions comprising aconductive oligomer covalently attached to a phosphoramidite nucleosideor to a solid support such as CPG, wherein said conductive oligomer hasthe formula:

[0033] The invention further provides compositions comprising anucleoside covalently linked to a metallocene.

[0034] The invention additionally provides composition comprising: (1)an electrode; (2) at least one metallocene; and (3) a conductiveoligomer covalently attached to both said electrode and saidmetallocene, wherein said conductive oligomer has the formula:

BRIEF DESCRIPTION OF THE DRAWINGS

[0035]FIG. 1 depicts the synthetic scheme for a conductive oligomercovalently attached to a uridine nucleoside via an amide bond.

[0036]FIG. 2 depicts the synthetic scheme for covalently attaching aconductive oligomer covalently attached to a uridine nucleoside via anamine bond.

[0037]FIG. 3 depicts the synthetic scheme for a conductive oligomercovalently attached to a uridine nucleoside via the base.

[0038]FIG. 4 depicts the synthetic scheme for a conductive oligomercovalently attached to a nucleoside via a phosphate of theribose-phosphate backbone. The conductive oligomer is a phenyl-acetyleneStructure 5 oligomer, although other oligomers may be used, andterminates in an ethyl pyridine protecting group, as described herein,for attachment to gold electrodes.

[0039]FIG. 5 depicts the synthetic scheme for a conductive oligomercovalently attached to a nucleoside via a phosphate of theribose-phosphate backbone, using an amide linkage and an ethylenelinker, although other linkers may be used. The conductive oligomer is aphenyl-acetylene Structure 5 oligomer, although other oligomers may beused, and terminates in an ethyl pyridine protecting group, as describedherein, for attachment to gold electrodes.

[0040]FIG. 6 depicts the synthetic scheme for a conductive polymercontaining an aromatic group with a substitution group. The conductiveoligomer is a phenyl-acetylene Structure 5 oligomer with a single methylR group on each phenyl ring, although other oligomers may be used, andterminates in an ethyl pyridine protecting group, as described herein,for attachment to gold electrodes.

[0041]FIG. 7 depicts the synthetic scheme for the synthesis of ametallocene, in this case ferrocene, linked via a conductive oligomer toan electrode. The conductive oligomer is a phenyl-acetylene Structure 5oligomer, although other oligomers may be used, and terminates in anethyl pyridine protecting group, as described herein, for attachment togold electrodes.

DETAILED DESCRIPTION OF THE INVENTION

[0042] The present invention capitalizes on the previous discovery thatelectron transfer apparently proceeds through the stacked π-orbitals ofthe heterocyclic bases of double stranded (hybridized) nucleic acid(“the π-way”). This finding allows the use of nucleic acids containingelectron transfer moieties to be used as nucleic acid probes. See PCTpublication WO 95/15971, hereby incorporated by reference in itsentirety, and cited references. This publication describes thesite-selective modification of nucleic acids with redox active moieties,i.e. electron donor and acceptor moieties, which allow the long-distanceelectron transfer through a double stranded nucleic acid. In general,electron transfer between electron donors and acceptors does not occurat an appreciable rate when the nucleic acid is single stranded, nordoes it occur appreciably unless nucleotide base pairing exists in thedouble stranded sequence between the electron donor and acceptor in thedouble helical structure. Thus, PCT publication WO 95/15971 and thepresent invention are directed to the use of nucleic acids with electrontransfer moieties, including electrodes, as probes for the detection oftarget sequences within a sample.

[0043] In one embodiment, the present invention provides for novel geneprobes, which are useful in molecular biology and diagnostic medicine.In this embodiment, single stranded nucleic acids having a predeterminedsequence and covalently attached electron transfer moieties, includingan electrode, are synthesized. The sequence is selected based upon aknown target sequence, such that if hybridization to a complementarytarget sequence occurs in the region between the electron donor and theelectron acceptor, electron transfer proceeds at an appreciable anddetectable rate. Thus, the invention has broad general use, as a newform of labelled gene probe. In addition, since detectable electrontransfer in unhybridized probes is not appreciable, the probes of thepresent invention allow detection of target sequences without theremoval of unhybridized probe. Thus, the invention is uniquely suited toautomated gene probe assays or field testing.

[0044] The present invention provides improved compositions comprisingnucleic acids covalently attached via conductive oligomers to anelectrode, of a general structure depicted below in Structure 1:

[0045] In Structure 1, the hatched marks on the left represent anelectrode. X is a conductive oligomer as defined herein. F₁ is a linkagethat allows the covalent attachment of the electrode and the conductiveoligomer, including bonds, atoms or linkers such as is described herein,for example as “A”, defined below. F₂ is a linkage that allows thecovalent attachment of the conductive oligomer to the nucleic acid, andmay be a bond, an atom or a linkage as is herein described. F₂ may bepart of the conductive oligomer, part of the nucleic acid, or exogeneousto both, for example, as defined herein for “Z”.

[0046] By “nucleic acid” or “oligonucleotide” or grammatical equivalentsherein means at least two nucleotides covalently linked together. Anucleic acid of the present invention will generally containphosphodiester bonds, although in some cases, as outlined below, anucleic acid analogs are included that may have alternate backbones,comprising, for example, phosphoramide (Beaucage et al., Tetrahedron49(10):1925 (1993) and references therein; Letsinger, J. Org. Chem.35:3800 (1970); Sprinzl et al., Eur. J. Biochem. 81:579 (1977);Letsinger et al., Nucl. Acids Res. 14:3487 (1986); Sawai et al, Chem.Lett. 805 (1984), Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988);and Pauwels et al., Chemica Scripta 26:141 91986)), phosphorothioate,phosphorodithioate, O-methylphophoroamidite linkages (see Eckstein,Oligonucleotides and Analogues: A Practical Approach, Oxford UniversityPress), and peptide nucleic acid backbones and linkages (see Egholm, J.Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed. Engl.31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et al., Nature380:207 (1996), all of which are incorporated by reference). Nucleicacids containing one or more carbocyclic sugars are also included withinthe definition of nucleic acids (see Jenkins et al., Chem. Soc. Rev.(1995) pp 169-176). These modifications of the ribose-phosphate backbonemay be done to facilitate the addition of electron transfer moieties, orto increase the stability and half-life of such molecules inphysiological environments.

[0047] Particularly preferred are peptide nucleic acids (PNA). Thesebackbones are substantially non-ionic under neutral conditions, incontrast to the highly charged phosphodiester backbone of naturallyoccurring nucleic acids. This results in two advantages. First, the PNAbackbone exhibits improved hybridization kinetics. PNAs have largerchanges in the melting temperature (Tm) for mismatched versus perfectlymatched basepairs. DNA and RNA typically exhibit a 2-4° C. drop in Tmfor an internal mismatch. With the non-ionic NA backbone, the drop iscloser to 7-9° C. This allows for better detection of mismatches.Similarly, due to their non-ionic nature, hybridization of the basesattached to these backbones is relatively insensitive to saltconcentration. This is particularly advantageous in the systems of thepresent invention, as a reduced salt hybridization solution has a lowerFaradaic current than a physiological salt solution (in the range of 150mM).

[0048] The nucleic acids may be single stranded or double stranded, asspecified, or contain portions of both double stranded or singlestranded sequence. The nucleic acid may be DNA, both genomic and cDNA,RNA or a hybrid, where the nucleic acid contains any combination ofdeoxyribo- and ribo-nucleotides, and any combination of bases, includinguracil, adenine, thymine, cytosine, guanine, inosine, xathanine andhypoxathanine, etc. As used herein, the term “nucleoside” includesnucleotides, and modified nucleosides such as amino modifiednucleosides.

[0049] The nucleosides and nucleic acids are covalently attached to aconductive oligomer. By “conductive oligomer” herein is meant asubstantially conducting oligomer, preferably linear, some embodimentsof which are referred to in the literature as “molecular wires”. By“substantially conducting” herein is meant that the rate of electrontransfer through the conductive oligomer is faster than the rate ofelectron transfer through single stranded nucleic acid, such that theconductive oligomer is not the rate limiting step in the detection ofhybridization. Stated differently, the resistance of the conductiveoligomer is less than that of the nucleic acid. Preferably, the rate ofelectron transfer through the conductive oligomer is faster than therate of electron transfer through double stranded nucleic acid, i.e.through the stacked π-orbitals of the double helix. Generally, theconductive oligomer has substantially overlapping π-orbitals, i.e.conjugated n-orbitals, as between the monomeric units of the conductiveoligomer, although the conductive oligomer may also contain one or moresigma (σ) bonds. Additionally, a conductive oligomer may be definedfunctionally by its ability to inject or receive electrons into or froman attached nucleic acid. Furthermore, the conductive oligomer is moreconductive than the insulators as defined herein.

[0050] In a preferred embodiment, the conductive oligomers have aconductivity, S, of from between about 10⁻⁶ to about 10⁴ Ω⁻¹cm⁻¹, withfrom about 10⁻⁵ to about 10³ Ω⁻¹cm⁻¹ being preferred, with these Svalues being calculated for molecules ranging from about 20 Å to about200 Å. As described below, insulators have a conductivity S of about10⁻⁷ Ω⁻¹cm⁻¹ or lower, with less than about 10⁻⁸ Ω⁻¹ cm⁻¹ beingpreferred. See generally Gardner et al., Sensors and Actuators A 51(1995) 57-66, incorporated herein by reference.

[0051] Desired characteristics of a conductive oligomer include highconductivity, sufficient solubility in organic solvents and/or water forsynthesis and use of the compositions of the invention, and preferablychemical resistance to reactions that occur i) during nucleic acidsynthesis (such that nucleosides containing the conductive oligomers maybe added to a nucleic acid synthesizer during the synthesis of thecompositions of the invention), ii) during the attachment of theconductive oligomer to an electrode, or iii) during hybridizationassays.

[0052] The oligomers of the invention comprise at least two monomericsubunits, as described herein. As is described more fully below,oligomers include homo- and hetero-oligomers, and include polymers.

[0053] In a preferred embodiment, the conductive oligomer has thestructure depicted in Structure 2:

[0054] As will be understood by those in the art, all of the structuresdepicted herein may have additional atoms or structures; i.e. theconductive oligomer of Structure 2 may be attached to electron transfermoieties, such as electrodes, transition metal complexes, organicelectron transfer moieties, and metallocenes, and to nucleic acids, orto several of these. Unless otherwise noted, the conductive oligomersdepicted herein will be attached at the left side to an electrode; thatis, as depicted in Structure 2, the left “Y” is connected to theelectrode as described herein and the right “Y”, if present, is attachedto the nucleic acid, either directly or through the use of a linker, asis described herein.

[0055] In this embodiment, Y is an aromatic group, n is an integer from1 to 50, g is either 1 or zero, e is an integer from zero to 10, and mis zero or 1. When g is 1, B-D is a conjugated bond, preferably selectedfrom acetylene, alkene, substituted alkene, amide, azo, —C═N— (including—N═C—, —CR═N— and —N═CR—), —Si═Si—, and —Si═C— (including —C═Si—,—Si═CR— and —CR═Si—). When g is zero, e is preferably 1, D is preferablycarbonyl, or a heteroatom moiety, wherein the heteroatom is selectedfrom oxygen, sulfur, nitrogen or phosphorus. Thus, suitable heteroatommoieties include, but are not limited to, —NH and —NR, wherein R is asdefined herein; substituted sulfur; sulfonyl (—SO₂—) sulfoxide (—SO—);phosphine oxide (—PO— and —RPO—); and thiophosphine (—PS— and —RPS—).However, when the conductive oligomer is to be attached to a goldelectrode, as outlined below, sulfur derivatives are not preferred.

[0056] By “aromatic group” or grammatical equivalents herein is meant anaromatic monocyclic or polycyclic hydrocarbon moiety generallycontaining 5 to 14 carbon atoms (although larger polycyclic ringsstructures may be made) and any carbocylic ketone or thioketonederivative thereof, wherein the carbon atom with the free valence is amember of an aromatic ring. Aromatic groups include arylene groups andaromatic groups with more than two atoms removed. For the purposes ofthis application aromatic includes heterocycle. “Heterocycle” or“heteroaryl” means an aromatic group wherein 1 to 5 of the indicatedcarbon atoms are replaced by a heteroatom chosen from nitrogen, oxygen,sulfur, phosphorus, boron and silicon wherein the atom with the freevalence is a member of an aromatic ring, and any heterocyclic ketone andthioketone derivative thereof. Thus, heterocycle includes thienyl,furyl, pyrrolyl, pyrimidinyl, oxalyl, indolyl, purinyl, quinolyl,isoquinolyl, thiazolyl, imidozyl, etc.

[0057] Importantly, the Y aromatic groups of the conductive oligomer maybe different, i.e. the conductive oligomer may be a heterooligomer. Thatis, a conductive oligomer may comprise a oligomer of a single type of Ygroups, or of multiple types of Y groups. Thus, in a preferredembodiment, when a barrier monolayer is used as is described below, oneor more types of Y groups are used in the conductive oligomer within themonolayer with a second type(s) of Y group used above the monolayerlevel. Thus, as is described herein, the conductive oligomer maycomprise Y groups that have good packing efficiency within the monolayerat the electrode surface, and a second type(s) of Y groups with greaterflexibility and hydrophilicity above the monolayer level to facilitatenucleic acid hybridization. For example, unsubstituted benzyl rings maycomprise the Y rings for monolayer packing, and substituted benzyl ringsmay be used above the monolayer. Alternatively, heterocylic rings,either substituted or unsubstituted, may be used above the monolayer.Additionally, in one embodiment, heterooligomers are used even when theconductive oligomer does not extend out of the monolayer.

[0058] The aromatic group may be substituted with a substitution group,generally depicted herein as R. R groups may be added as necessary toaffect the packing of the conductive oligomers, i.e. when the nucleicacids attached to the conductive oligomers form a monolayer on theelectrode, R groups may be used to alter the association of theoligomers in the monolayer. R groups may also be added to 1) alter thesolubility of the oligomer or of compositions containing the oligomers;2) alter the conjugation or electrochemical potential of the system; and3) alter the charge or characteristics at the surface of the monolayer.

[0059] Suitable R groups include, but are not limited to, hydrogen,alkyl, alcohol, aromatic, amino, amido, nitro, ethers, esters,aldehydes, sulfonyl, silicon moieties, halogens, sulfur containingmoieties, phosphorus containing moieties, and ethylene glycols. In thestructures depicted herein, R is hydrogen when the position isunsubstituted. It should be noted that some positions may allow twosubstitution groups, R and R′, in which case the R and R′ groups may beeither the same or different.

[0060] By “alkyl group” or grammatical equivalents herein is meant astraight or branched chain alkyl group, with straight chain alkyl groupsbeing preferred. If branched, it may be branched at one or morepositions, and unless specified, at any position. The alkyl group mayrange from about 1 to about 30 carbon atoms (C1-C30), with a preferredembodiment utilizing from about 1 to about 20 carbon atoms (C1-C20),with about C1 through about C12 to about C15 being preferred, and C1 toC5 being particularly preferred, although in some embodiments the alkylgroup may be much larger. Also included within the definition of analkyl group are cycloalkyl groups such as C5 and C6 rings, andheterocyclic rings with nitrogen, oxygen, sulfur or phosphorus. Alkylalso includes heteroalkyl, with heteroatoms of sulfur, oxygen, nitrogen,and silicone being preferred. Alkyl includes substituted alkyl groups.By “substituted alkyl group” herein is meant an alkyl group furthercomprising one or more substitution moieties “R”, as defined above.

[0061] By “amino groups” or grammatical equivalents herein is meant—NH₂, —NHR and —NR₂ groups, with R being as defined herein.

[0062] By “nitro group” herein is meant an —NO₂ group.

[0063] By “sulfur containing moieties” herein is meant compoundscontaining sulfur atoms, including but not limited to, thia-, thio- andsulfo-compounds, thiols (—SH and —SR), and sulfides (—RSR—). By“phosphorus containing moieties” herein is meant compounds containingphosphorus, including, but not limited to, phosphines and phosphates. By“silicon containing moieties” herein is meant compounds containingsilicon.

[0064] By “ether” herein is meant an —O—R group.

[0065] By “ester” herein is meant a —COOR group.

[0066] By “halogen” herein is meant bromine, iodine, chlorine, orfluorine. Preferred substituted alkyls are partially or fullyhalogenated alkyls such as CF₃, etc.

[0067] By “aldehyde” herein is meant —RCOH groups.

[0068] By “alcohol” herein is meant —OH groups, and alkyl alcohols —ROH.

[0069] By “amido” herein is meant —RCONH— or RCONR— groups.

[0070] By “ethylene glycol” herein is meant a —(O—CH₂—CH₂)_(n)— group,although each carbon atom of the ethylene group may also be singly ordoubly substituted, i.e. —(O—CR₂—CR₂)_(n)—, with R as described above.Ethylene glycol derivatives with other heteroatoms in place of oxygen(i.e. —(N—CH₂—CH₂)_(n)— or —(S—CH₂—CH₂)_(n)—, or with substitutiongroups) are also preferred.

[0071] Preferred substitution groups include, but are not limited to,methyl, ethyl, propyl, and ethylene glycol and derivatives thereof.

[0072] Preferred aromatic groups include, but are not limited to,phenyl, naphthyl, naphthalene, anthracene, phenanthroline, pyrole,pyridine, thiophene, porphyrins, and substituted derivatives of each ofthese, included fused ring derivatives.

[0073] In the conductive oligomers depicted herein, when g is 1, B-D isa bond linking two atoms or chemical moieties. In a preferredembodiment, B-D is a conjugated bond, containing overlapping orconjugated π-orbitals.

[0074] Preferred B-D bonds are selected from acetylene (—C≡C—, alsocalled alkyne or ethyne), alkene (—CH═CH—, also called ethylene),substituted alkene (—CR═CR—, —CH═CR— and —CR═CH—), amide (—NH—CO— and—NR—CO— or —CO—NH— and —CO—NR—), azo (—N═N—), esters and thioesters(—CO—O—, —O—CO—, —CS—O— and —O—CS—) and other conjugated bonds such as(—CH═N—, —CR═N—, —N═CH— and —N═CR—), (—SiH═SiH—, —SiR═SiH—, —SiR═SiH—,and —SiR═SiR—), (—SiH═CH—, —SiR═CH—, —SiH═CR—, —SiR═CR—, —CH═SiH—,—CR═SiH—, —CH═SiR—, and —CR═SiR—). Particularly preferred B-D bonds areacetylene, alkene, amide, and substituted derivatives of these three,and azo. Especially preferred B-D bonds are acetylene, alkene and amide.The oligomer components attached to double bonds may be in the trans orcis conformation, or mixtures. Thus, either B or D may include carbon,nitrogen or silicon. The substitution groups are as defined as above forR.

[0075] When g=0 in the Structure 2 conductive oligomer, e is preferably1 and the D moiety may be carbonyl or a heteroatom moiety as definedabove.

[0076] As above for the Y rings, within any single conductive oligomer,the B-D bonds (or D moieties, when g=0) may be all the same, or at leastone may be different. For example, when m is zero, the terminal B-D bondmay be an amide bond, and the rest of the B-D bonds may be acetylenebonds. Generally, when amide bonds are present, as few amide bonds aspossible are preferable, but in some embodiments all the B-D bonds areamide bonds. Thus, as outlined above for the Y rings, one type of B-Dbond may be present in the conductive oligomer within a monolayer asdescribed below, and another type above the monolayer level, to givegreater flexibility for nucleic acid hybridization.

[0077] In the structures depicted herein, n is an integer from 1 to 50,although longer oligomers may also be used (see for example Schumm etal., Angew. Chem. Int. Ed. Engl. 1994 33(13):1360). Without being boundby theory, it appears that for efficient hybridization of nucleic acidson a surface, the hybridization should occur at a distance from thesurface, i.e. the kinetics of hybridization increase as a function ofthe distance from the surface, particularly for long oligonucleotides of200 to 300 basepairs. Accordingly, the length of the conductive oligomeris such that the closest nucleotide of the nucleic acid is positionedfrom about 6 Å to about 100 Å (although distances of up to 500 Å may beused) from the electrode surface, with from about 25 Å to about 60 Åbeing preferred. Accordingly, n will depend on the size of the aromaticgroup, but generally will be from about 1 to about 20, with from about 2to about 15 being preferred and from about 3 to about 10 beingespecially preferred.

[0078] In the structures depicted herein, m is either 0 or 1. That is,when m is 0, the conductive oligomer may terminate in the B-D bond or Dmoiety, i.e. the D atom is attached to the nucleic acid either directlyor via a linker. In some embodiments, for example when the conductiveoligomer is attached to a phosphate of the ribose-phosphate backbone ofa nucleic acid, there may be additional atoms, such as a linker,attached between the conductive oligomer and the nucleic acid.Additionally, as outlined below, the D atom may be the nitrogen atom ofthe amino-modified ribose. Alternatively, when m is 1, the conductiveoligomer may terminate in Y, an aromatic group, i.e. the aromatic groupis attached to the nucleic acid or linker.

[0079] As will be appreciated by those in the art, a large number ofpossible conductive oligomers may be utilized. These include conductiveoligomers falling within the Structure 2 and Structure 9 formulas, aswell as other conductive oligomers, as are generally known in the art,including for example, compounds comprising fused aromatic rings orTeflon®-like oligomers, such as —(CF₂)_(n)—, —(CHF)_(n)— and—(CFR)_(n)—. See for example, Schumm et al., angew. Chem. Intl. Ed.Engl. 33:1361 (1994); Grosshenny et al., Platinum Metals Rev.40(1):26-35 (1996); Tour, Chem. Rev. 96:537-553 (1996); Hsung et al.,Organometallics 14:4808-4815 (1995; and references cited therein, all ofwhich are expressly incorporated by reference.

[0080] Particularly preferred conductive oligomers of this embodimentare depicted below:

[0081] Structure 3 is Structure 2 when g is 1. Preferred embodiments ofStructure 3 include: e is zero, Y is pyrole or substituted pyrole; e iszero, Y is thiophene or substituted thiophene; e is zero, Y is furan orsubstituted furan; e is zero, Y is phenyl or substituted phenyl; e iszero, Y is pyridine or substituted pyridine; e is 1, B-D is acetyleneand Y is phenyl or substituted phenyl (see Structure 5 below). Apreferred embodiment of Structure 3 is also when e is one, depicted asStructure 4 below:

[0082] Preferred embodiments of Structure 4 are: Y is phenyl orsubstituted phenyl and B-D is azo; Y is phenyl or substituted phenyl andB-D is alkene; Y is pyridine or substituted pyridine and B-D isacetylene; Y is thiophene or substituted thiophene and B-D is acetylene;Y is furan or substituted furan and B-D is acetylene; Y is thiophene orfuran (or substituted thiophene or furan) and B-D are alternating alkeneand acetylene bonds.

[0083] Most of the structures depicted herein utilize a Structure 4conductive oligomer. However, any Structure 4 oligomers may besubstituted with a Structure 2, 3 or 9 oligomer, or other conductingoligomer, and the use of such Structure 4 depiction is not meant tolimit the scope of the invention.

[0084] Particularly preferred embodiments of Structure 4 includeStructures 5, 6, 7 and 8, depicted below:

[0085] Particularly preferred embodiments of Structure 5 include: n istwo, m is one, and R is hydrogen; n is three, m is zero, and R ishydrogen; and the use of R groups to increase solubility.

[0086] When the B-D bond is an amide bond, as in Structure 6, theconductive oligomers are pseudopeptide oligomers. Although the amidebond in Structure 6 is depicted with the carbonyl to the left, i.e.—CONH—, the reverse may also be used, i.e. —NHCO—. Particularlypreferred embodiments of Structure 6 include: n is two, m is one, and Ris hydrogen; n is three, m is zero, and R is hydrogen (in thisembodiment, the terminal nitrogen (the D atom) may be the nitrogen ofthe amino-modified ribose); and the use of R groups to increasesolubility.

[0087] Preferred embodiments of Structure 7 include the first n is two,second n is one, m is zero, and all R groups are hydrogen, or the use ofR groups to increase solubility.

[0088] Preferred embodiments of Structure 8 include: the first n isthree, the second n is from 1-3, with m being either 0 or 1, and the useof R groups to increase solubility.

[0089] In a preferred embodiment, the conductive oligomer has thestructure depicted in Structure 9:

[0090] In this embodiment, C are carbon atoms, n is an integer from 1 to50, m is 0 or 1, J is a heteroatom selected from the group consisting ofnitrogen, silicon, phosphorus, sulfur, carbonyl or sulfoxide, and G is abond selected from alkane, alkene or acetylene, such that together withthe two carbon atoms the C-G-C group is an alkene (—CH═CH—), substitutedalkene (—CR═CR—) or mixtures thereof (—CH═CR— or —CR═CH—), acetylene(—C═C—), or alkane (—CR₂—CR₂—, with R being either hydrogen or asubstitution group as described herein). The G bond of each subunit maybe the same or different than the G bonds of other subunits; that is,alternating oligomers of alkene and acetylene bonds could be used, etc.However, when G is an alkane bond, the number of alkane bonds in theoligomer should be kept to a minimum, with about six or less sigma bondsper conductive oligomer being preferred. Alkene bonds are preferred, andare generally depicted herein, although alkane and acetylene bonds maybe substituted in any structure or embodiment described herein as willbe appreciated by those in the art.

[0091] In a preferred embodiment, the m of Structure 9 is zero. In aparticularly preferred embodiment, m is zero and G is an alkene bond, asis depicted in Structure 10:

[0092] The alkene oligomer of structure 10, and others depicted herein,are generally depicted in the preferred trans configuration, althougholigomers of cis or mixtures of trans and cis may also be used. Asabove, R groups may be added to alter the packing of the compositions onan electrode, the hydrophilicity or hydrophobicity of the oligomer, andthe flexibility, i.e. the rotational, torsional or longitudinalflexibility of the oligomer. n is as defined above.

[0093] In a preferred embodiment, R is hydrogen, although R may be alsoalkyl groups and polyethylene glycols or derivatives.

[0094] In an alternative embodiment, the conductive oligomer may be amixture of different types of oligomers, for example of structures 2 and9.

[0095] The conductive oligomers are covalently attached to the nucleicacids. By “covalently attached” herein is meant that two moieties areattached by at least one bond, including sigma bonds, pi bonds andcoordination bonds.

[0096] The nucleic acid is covalently attached to the conductiveoligomer, and the conductive oligomer is also covalently attached to theelectrode. In general, the covalent attachments are done in such amanner as to minimize the amount of unconjugated sigma bonds an electronmust travel from the electron donor to the electron acceptor. Thus,linkers are generally short, or contain conjugated bonds with few sigmabonds.

[0097] The covalent attachment of the nucleic acid and the conductiveoligomer may be accomplished in several ways. In a preferred embodiment,the attachment is via attachment to the base of the nucleoside, viaattachment to the backbone of the nucleic acid, or via a transitionmetal ligand, as described below. The techniques outlined below aregenerally described for naturally occuring nucleic acids, although aswill be appreciated by those in the art, similar techniques may be usedwith nucleic acid analogs.

[0098] In a preferred embodiment, the conductive oligomer is attached tothe base of a nucleoside of the nucleic acid. This may be done inseveral ways, depending on the oligomer, as is described below. In oneembodiment, the oligomer is attached to a terminal nucleoside, i.e.either the 3′ or 5′ nucleoside of the nucleic acid. Alternatively, theconductive oligomer is attached to an internal nucleoside.

[0099] The point of attachment to the base will vary with the base.While attachment at any position is possible, it is preferred to attachat positions not involved in hydrogen bonding to the complementary base.Thus, for example, generally attachment is to the 5 or 6 position ofpyrimidines such as uridine, cytosine and thymine. For purines such asadenine and guanine, the linkage is preferably via the 8 position.Attachment to non-standard bases is preferably done at the comparablepositions.

[0100] In one embodiment, the attachment is direct; that is, there areno intervening atoms between the conductive oligomer and the base. Inthis embodiment, for example, conductive oligomers with terminalacetylene bonds are attached directly to the base. Structure 11 is anexample of this linkage, using a Structure 4 conductive oligomer anduridine as the base, although other bases and conductive oligomers canbe used as will be appreciated by those in the art:

[0101] It should be noted that the pentose structures depicted hereinmay have hydrogen, hydroxy, phosphates or other groups such as aminogroups attached. In addition, the pentose and nucleoside structuresdepicted herein are depicted non-conventionally, as mirror images of thenormal rendering.

[0102] In addition, the base may contain additional modifications asneeded, i.e. the carbonyl or amine groups may be altered or protected,for example as is depicted in FIG. 3.

[0103] In an alternative embodiment, the attachment is through an amidebond using a linker as needed, as is generally depicted in Structure 12using uridine as the base and a Structure 4 oligomer:

[0104] Preferred embodiments of Structure 12 include Z is a methylene orethylene. The amide attachment can also be done using an amino group ofthe base, either a naturally occurring amino group such as in cytidineor adenidine, or from an amino-modified base as are known in the art.

[0105] In this embodiment, Z is a linker. Preferably, Z is a shortlinker of about 1 to about 5 atoms, that may or may not contain alkenebonds. Linkers are known in the art; for example, homo-orhetero-bifunctional linkers as are well known (see 1994 Pierce ChemicalCompany catalog, technical section on cross-linkers, pages 155-200,incorporated herein by reference). Preferred Z linkers include, but arenot limited to, alkyl groups and alkyl groups containing heteroatommoieties, with short alkyl groups, esters, epoxy groups and ethyleneglycol and derivatives being preferred, with propyl, acetylene, and C₂alkene being especially preferred.

[0106] In a preferred embodiment, the attachment of the nucleic acid andthe conductive oligomer is done via attachment to the backbone of thenucleic acid.

[0107] This may be done in a number of ways, including attachment to aribose of the ribose-phosphate backbone, or to the phosphate of thebackbone, or other groups of analogous backbones.

[0108] As a preliminary matter, it should be understood that the site ofattachment in this embodiment may be to a 3′ or 5′ terminal nucleotide,or to an internal nucleotide, as is more fully described below.

[0109] In a preferred embodiment, the conductive oligomer is attached tothe ribose of the ribose-phosphate backbone. This may be done in severalways. As is known in the art, nucleosides that are modified at eitherthe 2′ or 3′ position of the ribose with amino groups, sulfur groups,silicone groups, phosphorus groups, or oxo groups can be made (Imazawaet al., J. Org. Chem., 44:2039 (1979); Hobbs et al., J. Org. Chem.42(4):714 (1977); Verheyden et al., J. Orrg. Chem. 36(2):250 (1971);McGee et al., J. Org. Chem. 61:781-785 (1996); Mikhailopulo et al.,Liebigs. Ann. Chem. 513-519 (1993); McGee et al., Nucleosides &Nucleotides 14(6):1329 (1995), all of which are incorporated byreference). These modified nucleosides are then used to add theconductive oligomers.

[0110] A preferred embodiment utilizes amino-modified nucleosides. Theseamino-modified riboses can then be used to form either amide or aminelinkages to the conductive oligomers. In a preferred embodiment, theamino group is attached directly to the ribose, although as will beappreciated by those in the art, short linkers such as those describedherein for “Z” may be present between the amino group and the ribose.

[0111] In a preferred embodiment, an amide linkage is used forattachment to the ribose. Preferably, if the conductive oligomer ofStructures 2-4 is used, m is zero and thus the conductive oligomerterminates in the amide bond. In this embodiment, the nitrogen of theamino group of the amino-modified ribose is the “D” atom of theconductive oligomer. Thus, a preferred attachment of this embodiment isdepicted in Structure 13 (using the Structure 4 conductive oligomer):

[0112] As will be appreciated by those in the art, Structure 13 has theterminal bond fixed as an amide bond.

[0113] In a preferred embodiment, a heteroatom linkage is used, i.e.oxo, amine, sulfur, etc. A preferred embodiment utilizes an aminelinkage. Again, as outlined above for the amide linkages, for aminelinkages, the nitrogen of the amino-modified ribose may be the “D” atomof the conductive oligomer when the Structure 4 conductive oligomer isused. Thus, for example, Structures 14 and 15 depict nucleosides withthe Structures 4 and 10 conductive oligomers, respectively, using thenitrogen as the heteroatom, athough other heteroatoms can be used:

[0114] In Structure 14, preferably both m and t are not zero.

[0115] In Structure 15, Z is as defined above. Suitable linkers includemethylene and ethylene.

[0116] In an alternative embodiment, the conductive oligomer iscovalently attached to the nucleic acid via the phosphate of theribose-phosphate backbone (or analog) of a nucleic acid. In thisembodiment, the attachment is direct, utilizes a linker or via an amidebond. Structure 16 depicts a direct linkage, and Structure 17 depictslinkage via an amide bond (both utilize the Structure 4 conductiveoligomer, although Structure 9 conductive oligomers are also possible).Structures 16 and 17 depict the conductive oligomer in the 3′ position,although the 5′ position is also possible. Furthermore, both Structures16 and 17 depict naturally occurring phosphodiester bonds, although asthose in the art will appreciate, non-standard analogs of phosphodiesterbonds may also be used.

[0117] In Structure 16, if the terminal Y is present (i.e. m=1), thenpreferably Z is not present (i.e. t=0). If the terminal Y is notpresent, then Z is preferably present.

[0118] Structure 17 depicts a preferred embodiment, wherein the terminalB-D bond is an amide bond, the terminal Y is not present, and Z is alinker, as defined herein.

[0119] In a preferred embodiment, the conductive oligomer is covalentlyattached to the nucleic acid via a transition metal ligand. In thisembodiment, the conductive oligomer is covalently attached to a ligandwhich provides one or more of the coordination atoms for a transitionmetal. In one embodiment, the ligand to which the conductive oligomer isattached also has the nucleic acid attached, as is generally depictedbelow in Structure 18. Alternatively, the conductive oligomer isattached to one ligand, and the nucleic acid is attached to anotherligand, as is generally depicted below in Structure 19. Thus, in thepresence of the transition metal, the conductive oligomer is covalentlyattached to the nucleic acid. Both of these structures depict Structure4 conductive oligomers, although other oligomers may be utilized.Structures 18 and 19 depict two representative structures:

[0120] In the structures depicted herein, M is a metal atom, withtransition metals being preferred. Suitable transition metals for use inthe invention include, but are not limited to, cadmium (Cd), copper(Cu), cobalt (Co), palladium (Pd), zinc (Zn), iron (Fe), ruthenium (Ru),rhodium (Rh), osmium (Os), rhenium (Re), platinium (Pt), scandium (Sc),titanium (Ti), Vanadium (V), chromium (Cr), manganese (Mn), nickel (Ni),Molybdenum (Mo), technetium (Tc), tungsten (W), and iridium (Ir). Thatis, the first series of transition metals, the platinum metals (Ru, Rh,Pd, Os, Ir and Pt), along with Fe, Re, W, Mo and Tc, are preferred.Particularly preferred are ruthenium, rhenium, osmium, platinium andiron.

[0121] L are the co-ligands, that provide the coordination atoms for thebinding of the metal ion. As will be appreciated by those in the art,the number and nature of the co-ligands will depend on the coordinationnumber of the metal ion. Mono- di- or polydentate co-ligands may be usedat any position. Thus, for example, when the metal has a coordinationnumber of six, the L from the terminus of the conductive oligomer, the Lcontributed from the nucleic acid, and r, add up to six. Thus, when themetal has a coordination number of six, r may range from zero (when allcoordination atoms are provided by the other two ligands) to four, whenall the co-ligands are monodentate. Thus generally, r will be from 0 to8, depending on the coordination number of the metal ion and the choiceof the other ligands.

[0122] In one embodiment, the metal ion has a coordination number of sixand both the ligand attached to the conductive oligomer and the ligandattached to the nucleic acid are at least bidentate; that is, r ispreferably zero, one (i.e. the remaining co-ligand is bidentate) or two(two monodentate co-ligands are used).

[0123] As will be appreciated in the art, the co-ligands can be the sameor different. Suitable ligands fall into two categories: ligands whichuse nitrogen, oxygen, sulfur, carbon or phosphorus atoms (depending onthe metal ion) as the coordination atoms (generally referred to in theliterature as sigma (σ) donors) and organometallic ligands such asmetallocene ligands (generally referred to in the literature as pi (π)donors, and depicted herein as L_(m)). Suitable nitrogen donatingligands are well known in the art and include, but are not limited to,NH₂; NHR; NRR′; pyridine; pyrazine; isonicotinamide; imidazole;bipyridine and substituted derivatives of bipyridine; terpyridine andsubstituted derivatives; phenanthrolines, particularly1,10-phenanthroline (abbreviated phen) and substituted derivatives ofphenanthrolines such as 4,7-dimethylphenanthroline anddipyridol[3,2-a:2′,3′-c]phenazine (abbreviated dppz); dipyridophenazine;1,4,5,8,9,12-hexaazatriphenylene (abbreviated hat);9,10-phenanthrenequinone diimine (abbreviated phi);1,4,5,8-tetraazaphenanthrene (abbreviated tap);1,4,8,11-tetra-azacyclotetradecane (abbreviated cyclam) and isocyanide.Substituted derivatives, including fused derivatives, may also be used.In some embodiments, porphyrins and substituted derivatives of theporphyrin family may be used. See for example, ComprehensiveCoordination Chemistry, Ed. Wilkinson et al., Pergammon Press, 1987,Chapters 13.2 (pp73-98), 21.1 (pp. 813-898) and 21.3 (pp 915-957), allof which are hereby expressly incorporated by reference.

[0124] Suitable sigma donating ligands using carbon, oxygen, sulfur andphosphorus are known in the art. For example, suitable sigma carbondonors are found in Cotton and Wilkenson, Advanced Organic Chemistry,5th Edition, John Wiley & Sons, 1988, hereby incorporated by reference;see page 38, for example. Similarly, suitable oxygen ligands includecrown ethers, water and others known in the art. Phosphines andsubstituted phosphines are also suitable; see page 38 of Cotton andWilkenson.

[0125] The oxygen, sulfur, phosphorus and nitrogen-donating ligands areattached in such a manner as to allow the heteroatoms to serve ascoordination atoms.

[0126] In a preferred embodiment, organometallic ligands are used. Inaddition to purely organic compounds for use as redox moieties, andvarious transition metal coordination complexes with δ-bonded organicligand with donor atoms as heterocyclic or exocyclic substituents, thereis available a wide variety of transition metal organometallic compoundswith π-bonded organic ligands (see Advanced Inorganic Chemistry, 5thEd., Cotton & Wilkinson, John Wiley & Sons, 1988, chapter 26;Organometallics, A Concise Introduction, Elschenbroich et al., 2nd Ed.,1992, VCH; and Comprehensive Organometallic Chemistry II, A Review ofthe Literature, 1982-1994, Abel et al. Ed., Vol. 7, chapters 7, 8, 10 &11, Pergamon Press, hereby expressly incorporated by reference). Suchorganometallic ligands include cyclic aromatic compounds such as thecyclopentadienide ion [C₅H₅(−1)] and various ring substituted and ringfused derivatives, such as the indenylide (−1) ion, that yield a classof bis(cyclopentadieyl)metal compounds, (i.e. the metallocenes); see forexample Robins et al., J. Am. Chem. Soc. 104:1882-1893 (1982); andGassman et al., J. Am. Chem. Soc. 108:4228-4229 (1986), incorporated byreference. Of these, ferrocene [(C₅H₅)₂Fe] and its derivatives areprototypical examples which have been used in a wide variety of chemical(Connelly et al., Chem. Rev. 96:877-910 (1996), incorporated byreference) and electrochemical (Geiger et al., Advances inOrganometallic Chemistry 23:1-93; and Geiger et al., Advances inOrganometallic Chemistry 24:87, incorporated by reference) electrontransfer or “redox” reactions. Metallocene derivatives of a variety ofthe first, second and third row transition metals are potentialcandidates as redox moieties that are covalently attached to either theribose ring or the nucleoside base of nucleic acid. Other potentiallysuitable organometallic ligands include cyclic arenes such as benzene,to yield bis(arene)metal compounds and their ring substituted and ringfused derivatives, of which bis(benzene)chromium is a prototypicalexample, Other acyclic π-bonded ligands such as the allyl(−1) ion, orbutadiene yield potentially suitable organometallic compounds, and allsuch ligands, in conjuction with other π-bondedand δ-bonded ligandsconstitute the general class of organometallic compounds in which thereis a metal to carbon bond. Electrochemical studies of various dimers andoligomers of such compounds with bridging organic ligands, andadditional non-bridging ligands, as well as with and without metal-metalbonds are potential candidate redox moieties in nucleic acid analysis.When one or more of the co-ligands is an organometallic ligand, theligand is generally attached via one of the carbon atoms of theorganometallic ligand, although attachment may be via other atoms forheterocyclic ligands. Preferred organometallic ligands includemetallocene ligands, including substituted derivatives and themetalloceneophanes (see page 1174 of Cotton and Wilkenson, supra).

[0127] As described herein, any combination of ligands may be used.Preferred combinations include: a) all ligands are nitrogen donatingligands; b) all ligands are organometallic ligands; and c) the ligand atthe terminus of the conductive oligomer is a metallocene ligand and theligand provided by the nucleic acid is a nitrogen donating ligand, withthe other ligands, if needed, are either nitrogen donating ligands ormetallocene ligands, or a mixture. These combinations are depicted inrepresentative structures using the conductive oligomer of Structure 4are depicted in Structures 20 (using phenanthroline and amino asrepresentative ligands), 21 (using ferrocene as the metal-ligandcombination) and 22 (using cyclopentadienyl and amino as representativeligands).

[0128] In a preferred embodiment, the ligands used in the invention showaltered fluorescent properties depending on the redox state of thechelated metal ion. As described below, this thus serves as anadditional mode of detection of electron transfer through nucleic acid.

[0129] In a preferred embodiment, as is described more fully below, theligand attached to the nucleic acid is an amino group attached to the 2′or 3′ position of a ribose of the ribose-phosphate backbone. This ligandmay contain a multiplicity of amino groups so as to form a polydentateligand which binds the metal ion. Other preferred ligands includecyclopentadiene and phenanthroline.

[0130] As described herein, the compositions described herein ofnucleosides covalently attached to conductive oligomers may beincorporated into a longer nucleic acid at any number of positions,including either the 5′ or 3′ terminus of the nucleic acid or anyinternal position. As is outlined below, this is generally done byadding a nucleotide with a covalently attached conductive oligomer to anoligonucleotide synthetic reaction at any position. After synthesis iscomplete, the nucleic acid with the covalently attached conductiveoligomer is attached to an electrode. Thus, any number of additionalnucleotides, modified or not, may be included at any position.Alternatively, the compositions are made via post-nucleic acid synthesismodifications.

[0131] The total length of the nucleic acid will depend on its use.Generally, the nucleic acid compositions of the invention are useful asoligonucleotide probes. As is appreciated by those in the art, thelength of the probe will vary with the length of the target sequence andthe hybridization and wash conditions. Generally, oligonucleotide probesrange from about 8 to about 50 nucleotides, with from about 10 to about30 being preferred and from about 12 to about 25 being especiallypreferred. In some cases, very long probes may be used, e.g. 50 to200-300 nucleotides in length.

[0132] Also of consideration is the distance between the nucleosidecontaining the electrode, i.e. a first electron transfer moiety, and thenucleoside containing a second electron transfer moiety. Electrontransfer proceeds between the two electron transfer moieties. Since therate of electron transfer is distance dependent, the distance betweenthe two electron transfer moieties preferably ranges from about 1 toabout 30 basepairs, with from about 3 to about 20 basepairs beingpreferred and from about 5 to about 15 basepairs being particularlypreferred and from about 5 to 10 being especially preferred. However,probe specificity can be increased by adding oligonucleotides on eitherside of the electron transfer moieties, thus increasing probespecificity without increasing the distance an electron must travel.

[0133] Thus, in the structures depicted herein, nucleosides may bereplaced with nucleic acids.

[0134] In a preferred embodiment, the conductive oligomers withcovalently attached nucleosides or nucleic acids as depicted herein arecovalently attached to an electrode. Thus, one end or terminus of theconductive oligomer is attached to the nucleoside or nucleic acid, andthe other is attached to an electrode. In some embodiments it may bedesirable to have the conductive oligomer attached at a position otherthan a terminus, or even to have a branched conductive oligomer that isattached to an electrode at one terminus and to two or more nucleosidesat other termini, although this is not preferred. Similarly, theconductive oligomer may be attached at two sites to the electrode.

[0135] By “electrode” herein is meant a composition, which, whenconnected to an electronic device, is able to sense a current or chargeand convert it to a signal. Thus, an electrode is an electron transfermoiety as described herein. Preferred electodes are known in the art andinclude, but are not limited to, certain metals and their oxides,including gold; platinum; palladium; silicon; aluminum; metal oxideelectrodes including platinum oxide, titanium oxide, tin oxide, indiumtin oxide, palladium oxide, silicon oxide, aluminum oxide, molybdenumoxide (Mo₂O₆), tungsten oxide (WO₃) and ruthenium oxides; and carbon(including glassy carbon electrodes, graphite and carbon paste).Preferred electrodes include gold, silicon, carbon and metal oxideelectrodes.

[0136] The electrodes described herein are depicted as a flat surface,which is only one of the possible conformations of the electrode and isfor schematic purposes only. The conformation of the electrode will varywith the detection method used. For example, flat planar electrodes maybe preferred for optical detection methods, or when arrays of nucleicacids are made, thus requiring addressable locations for both synthesisand detection. Alternatively, for single probe analysis, the electrodemay be in the form of a tube, with the conductive oligomers and nucleicacids bound to the inner surface. This allows a maximum of surface areacontaining the nucleic acids to be exposed to a small volume of sample.

[0137] The covalent attachment of the conductive oligomer containing thenucleoside may be accomplished in a variety of ways, depending on theelectrode and the conductive oligomer used. Generally, some type oflinker is used, as depicted below as “A” in Structure 23, where X is theconductive oligomer, and the hatched surface is the electrode:

[0138] In this embodiment, A is a linker or atom. The choice of “A” willdepend in part on the characteristics of the electrode. Thus, forexample, A may be a sulfur moiety when a gold electrode is used.Alternatively, when metal oxide electrodes are used, A may be a silicon(silane) moiety attached to the oxygen of the oxide (see for exampleChen et al., Langmuir 10:3332-3337 (1994); Lenhard et al., J.Electroanal. Chem. 78:195-201 (1977), both of which are expresslyincorporated by reference). When carbon based electrodes are used, A maybe an amino moiety (preferably a primary amine; see for exampleDeinhammer et al., Langmuir 10:1306-1313 (1994)). Thus, preferred Amoieties include, but are not limited to, silane moieties, sulfurmoieties (including alkyl sulfur moieties), and amino moieties. In apreferred embodiment, epoxide type linkages with redox polymers such asare known in the art are not used.

[0139] Although depicted herein as a single moiety, the conductiveoligomer may be attached to the electrode with more than one “A” moiety;the “A” moieties may be the same or different. Thus, for example, whenthe electrode is a gold electrode, and “A” is a sulfur atom or moiety,such as generally depicted below in Structure 27, multiple sulfur atomsmay be used to attach the conductive oligomer to the electrode, such asis generally depicted below in Structures 24, and 26. As will beappreciated by those in the art, other such structures can be made. InStructures 24, 25 and 26, the A moiety is just a sulfur atom, butsubstituted sulfur moieties may also be used.

[0140] It should also be noted that similar to Structure 26, it may bepossible to have a a conductive oligomer terminating in a single carbonatom with three sulfur moities attached to the electrode.

[0141] In a preferred embodiment, the electrode is a gold electrode, andattachment is via a sulfur linkage as is well known in the art, i.e. theA moiety is a sulfur atom or moiety. Although the exact characteristicsof the gold-sulfur attachment are not known, this linkage is consideredcovalent for the purposes of this invention. A representative structureis depicted in Structure 27. Structure 27 depicts the “A” linker ascomprising just a sulfur atom, although additional atoms may be present(i.e. linkers from the sulfur to the conductive oligomer or substitutiongroups).

[0142] In a preferred embodiment, the electrode is a carbon electrode,i.e. a glassy carbon electrode, and attachment is via a nitrogen of anamine group. A representative structure is depicted in Structure 28.Again, additional atoms may be present, i.e. Z type linkers.

[0143] In Structure 29, the oxygen atom is from the oxide of the metaloxide electrode. The Si atom may also contain other atoms, i.e. be asilicon moiety containing substitution groups.

[0144] Thus, in a preferred embodiment, electrodes are made thatcomprise conductive oligomers attached to nucleic acids for the purposesof hybridization assays, as is more fully described herein. As will beappreciated by those in the art, electrodes can be made that have asingle species of nucleic acid, i.e. a single nucleic acid sequence, ormultiple nucleic acid species.

[0145] In addition, as outlined herein, the use of a solid support suchas an electrode enables the use of these gene probes in an array form.The use of oligonucleotide arrays are well known in the art. Inaddition, techniques are known for “addressing” locations within anelectrode and for the surface modification of electrodes. Thus, in apreferred embodiment, arrays of different nucleic acids are laid down onthe electrode, each of which are covalently attached to the electrodevia a conductive linker. In this embodiment, the number of differentprobe species of oligonucleotides may vary widely, from one tothousands, with from about 4 to about 100,000 being preferred, and fromabout 10 to about 10,000 being particularly preferred.

[0146] In a preferred embodiment, the electrode further comprises apassavation agent, preferably in the form of a monolayer on theelectrode surface. As outlined above, the efficiency of oligonucleotidehybridization may increase when the oligonucleotide is at a distancefrom the electrode. A passavation agent layer facilitates themaintenance of the nucleic acid away from the electrode surface. Inaddition, a passavation agent serves to keep charge carriers away fromthe surface of the electrode. Thus, this layer helps to preventelectrical contact between the electrodes and the electron transfermoieties, or between the electrode and charged species within thesolvent. Such contact can result in a direct “short circuit” or anindirect short circuit via charged species which may be present in thesample. Accordingly, the monolayer of passavation agents is preferablytightly packed in a uniform layer on the electrode surface, such that aminimum of “holes” exist. Alternatively, the passavation agent may notbe in the form of a monolayer, but may be present to help the packing ofthe conductive oligomers or other characteristics.

[0147] The passavation agents thus serve as a physical barrier to blocksolvent accesibility to the electrode. As such, the passavation agentsthemselves may in fact be either (1) conducting or (2) nonconducting,i.e. insulating, molecules. Thus, in one embodiment, the passavationagents are conductive oligomers, as described herein, with or without aterminal group to block or decrease the transfer of charge to theelectrode. Other passavation agents which may be conductive includeoligomers of —(CF₂)_(n)—, —(CHF)_(n)— and —(CFR)_(n)—. In a preferredembodiment, the passavation agents are insulator moieties.

[0148] An “insulator” is a substantially nonconducting oligomer,preferably linear. By “substantially nonconducting” herein is meant thatthe rate of electron transfer through the insulator is slower than therate of electron transfer through the stacked π-orbitals of doublestranded nucleic acid. Stated differently, the electrical resistance ofthe insulator is higher than the electrical resistance of the nucleicacid. In a preferred embodiment, the rate of electron transfer throughthe insulator is slower than or comparable to the rate through singlestranded nucleic acid. Similarly, the rate of electron transfer throughthe insulator is preferrably slower than the rate through the conductiveoligomers described herein. It should be noted however, as outlined inthe Examples, that even oligomers generally considered to be insulators,such as —(CH₂)₁₆ molecules, still may transfer electrons, albeit at aslow rate.

[0149] In a preferred embodiment, the insulators have a conductivity, S,of about 10⁻⁷ Ω⁻¹cm⁻¹ or lower, with less than about 10⁻⁸ Ω⁻¹cm⁻¹ beingpreferred. See generally Gardner et al., supra.

[0150] Generally, insulators are alkyl or heteroalkyl oligomers ormoieties with sigma bonds, although any particular insulator moleculemay contain aromatic groups or one or more conjugated bonds. By“heteroalkyl” herein is meant an alkyl group that has at least oneheteroatom, i.e. nitrogen, oxygen, sulfur, phosphorus, silicon or boronincluded in the chain. Alternatively, the insulator may be quite similarto a conductive oligomer with the addition of one or more heteroatoms orbonds that serve to inhibit or slow, preferably substantially, electrontransfer.

[0151] The passavation agents, including insulators, may be substitutedwith R groups as defined herein to alter the packing of the moieties orconductive oligomers on an electrode, the hydrophilicity orhydrophobicity of the insulator, and the flexibility, i.e. therotational, torsional or longitudinal flexibility of the insulator. Forexample, branched alkyl groups may be used. In addition, the terminus ofthe passavation agent, including insulators, may contain an additionalgroup to influence the exposed surface of the monolayer. For example,there may be negatively charged groups on the terminus to form anegatively charged surface such that when the nucleic acid is DNA or RNAthe nucleic acid is repelled or prevented from lying down on thesurface, to facilitate hybridization.

[0152] The length of the passavation agent will vary as needed. Asoutlined above, it appears that hybridization is more efficient at adistance from the surface. Thus, the length of the passavation agents issimilar to the length of the conductive oligomers, as outlined above. Inaddition, the conductive oligomers may be basically the same length asthe passavation agents or longer than them, resulting in the nucleicacids being more accessible to the solvent for hybridization.

[0153] The monolayer may comprise a single type of passavation agent,including insulators, or different types.

[0154] Suitable insulators are known in the art, and include, but arenot limited to, —(CH₂)_(n)—, —(CRH)_(n)—, and —(CR₂)_(n)—, ethyleneglycol or derivatives using other heteroatoms in place of oxygen, i.e.nitrogen or sulfur (sulfur derivatives are not preferred when theelectrode is gold).

[0155] The passavation agents are generally attached to the electrode inthe same manner as the conductive oligomer, and may use the same “A”linker as defined above.

[0156] In a preferred embodiment, the compositions of the presentinvention comprise a conductive oligomer, covalently attached to both anelectrode, which serves as a first electron transfer moiety, and anucleic acid. In this embodiment, the conductive oligomer preferably hasthe structure depicted in Structures 2, 3, 4, 9 or 10.

[0157] In a preferred embodiment, the compositions of the presentinvention comprise a conductive oligomer, covalently attached to both anelectrode, which serves as a first electron transfer moiety, and anucleic acid, which has at least a second covalently attached electrontransfer moiety.

[0158] In one embodiment, a nucleic acid is modified with more than twoelectron transfer moieties. For example, to increase the signal obtainedfrom the probe, or alter the required detector sensitivity, a pluralityof electron transfer moieties may be used. See PCT publication WO95/15971.

[0159] The terms “electron donor moiety”, “electron acceptor moiety”,and “electron transfer moieties” or grammatical equivalents hereinrefers to molecules capable of electron transfer under certainconditions. It is to be understood that electron donor and acceptorcapabilities are relative; that is, a molecule which can lose anelectron under certain experimental conditions will be able to accept anelectron under different experimental conditions. It is to be understoodthat the number of possible electron donor moieties and electronacceptor moieties is very large, and that one skilled in the art ofelectron transfer compounds will be able to utilize a number ofcompounds in the present invention. Preferred electron transfer moietiesinclude, but are not limited to, transition metal complexes, organicelectron transfer moieties, and electrodes.

[0160] In a preferred embodiment, the electron transfer moieties aretransition metal complexes. Transition metals are those whose atoms havea partial or complete d shell of electrons. Suitable transition metalsfor use in the invention are listed above.

[0161] The transition metals are complexed with a variety of ligands, L,defined above, to form suitable transition metal complexes, as is wellknown in the art.

[0162] In addition to transition metal complexes, other organic electrondonors and acceptors may be covalently attached to the nucleic acid foruse in the invention. These organic molecules include, but are notlimited to, riboflavin, xanthene dyes, azine dyes, acridine orange,N,N′-dimethyl-2,7-diazapyrenium dichloride (DAP²⁺), methylviologen,ethidium bromide, quinones such asN,N′-dimethylanthra(2,1,9-def-6,5,10-d′e′f′)diisoquinoline dichloride(ADIQ²⁺); porphyrins ([meso-tetrakis(N-methyl-x-pyridinium)porphyrintetrachloride], varlamine blue B hydrochloride, Bindschedler's green;2,6-dichloroindophenol, 2,6-dibromophenolindophenol; Brilliant crestblue (3-amino-9-dimethyl-amino-10-methylphenoxyazine chloride),methylene blue; Nile blue A (aminoaphthodiethylaminophenoxazinesulfate), indigo-5,5′,7,7′-tetrasulfonic acid, indigo-5,5′,7-trisulfonicacid; phenosafranine, indigo-5-monosulfonic acid; safranine T;bis(dimethylglyoximato)-iron(II) chloride; induline scarlet, neutralred, anthracene, coronene, pyrene, 9-phenylanthracene, rubrene,binaphthyl, DPA, phenothiazene, fluoranthene, phenanthrene, chrysene,1,8-diphenyl-1,3,5,7-octatetracene, naphthalene, acenaphthalene,perylene, TMPD and analogs and subsitituted derivatives of thesecompounds.

[0163] In one embodiment, the electron donors and acceptors are redoxproteins as are known in the art. However, redox proteins in manyembodiments are not preferred.

[0164] The choice of the specific electron transfer moieties will beinfluenced by the type of electron transfer detection used, as isgenerally outlined below.

[0165] In a preferred embodiment, these electron transfer moieties arecovalently attached to the nucleic acid in a variety of positions. In apreferred embodiment, the attachment is via attachment to the base ofthe nucleoside, or via attachment to the backbone of the nucleic acid,including either to a ribose of the ribose-phosphate backbone or to aphosphate moiety. In the preferred embodiments, the compositions of theinvention are designed such that the electron transfer moieties are asclose to the “π-way” as possible without significantly disturbing thesecondary and tertiary structure of the double helical nucleic acid,particularly the Watson-Crick basepairing. Alternatively, the attachmentcan be via a conductive oligomer, which is used as outlined above with anucleoside and an electrode; that is, an electron transfer moiety may becovalently attached to a conductive oligomer at one end and to anucleoside at the other, thus forming a general structure depicted inStructure 30:

[0166] In Structure 30, ETM is an electron transfer moiety, X is aconductive oligomer, and q is an integer from zero to about 25, withpreferred q being from about 4 to about 15. Additionally, linkermoieties, for example as are generally described herein as “Z”, may alsobe present between the nucleoside and the conductive oligomer, and/orbetween the, conductive oligomer and the electron transfer moiety. Thedepicted nucleosides may be either terminal or internal nucleosides, andare usually separated by a number of nucleosides.

[0167] In a preferred embodiment, the second electron transfer moiety isattached to the base of a nucleoside, as is generally outlined above forattachment of the conductive oligomer. This is preferably done to thebase of an internal nucleoside. Surprisingly and unexpectedly, thisattachment does not perturb the Watson-Crick basepairing of the base towhich the electron transfer moiety is attached, as long as the moiety isnot too large. In fact, it appears that attachment at this site actuallyresults in less perturbation than attachment at the ribose of theribose-phosphate backbone, as measured by nucleic acid melting curves.

[0168] Thus, when attachment to an internal base is done, the size ofthe second electron transfer moiety should be such that the structure ofdouble stranded nucleic acid containing the base-attached electrontransfer moiety is not significantly disrupted, and will not disrupt theannealing of single stranded nucleic acids. Preferrably, then, ligandsand full second electron transfer moieties are generally smaller thanthe size of the major groove of double stranded nucleic acid.

[0169] Alternatively, the second electron transfer moiety can beattached to the base of a terminal nucleoside. Thus, when the targetsequence to be detected is n nucleosides long, a probe can be made whichhas the second electron transfer moiety attached at the n base.Alternatively, the probe may contain an extra terminal nucleoside at anend of the nucleic acid (n+1 or n+2), which are used to covalentlyattach the electron transfer moieties but which do not participate inbasepair hybridization. Additionally, it is preferred that upon probehybridization, the terminal nucleoside containing the electron transfermoiety covalently attached at the base be directly adjacent toWatson-Crick basepaired nucleosides; that is, the electron transfermoiety should be as close as possible to the stacked π-orbitals of thebases such that an electron travels through a minimum of a bonds toreach the “π-way”, or alternatively can otherwise electronically contactthe π-way.

[0170] The covalent attachment to the base will depend in part on thesecond electron transfer moiety chosen, but in general is similar to theattachment of conductive oligomers to bases, as outlined above. In apreferred embodiment, the second electron transfer moiety is atransition metal complex, and thus attachment of a suitable metal ligandto the base leads to the covalent attachment of the electron transfermoiety. Alternatively, similar types of linkages may be used for theattachment of organic electron transfer moieties, as will be appreciatedby those in the art.

[0171] In one embodiment, the C4 attached amino group of cytosine, theC6 attached amino group of adenine, or the C2 attached amino group ofguanine may be used as a transition metal ligand, although in thisembodiment attachment at a terminal base is preferred since attachmentat these positions will perturb Watson-Crick basepairing.

[0172] Ligands containing aromatic groups can be attached via acetylenelinkages as is known in the art (see Comprehensive Organic Synthesis,Trost et al., Ed., Pergamon Press, Chapter 2.4: Coupling ReactionsBetween sp² and sp Carbon Centers, Sonogashira, pp521-549, andpp950-953, hereby incorporated by reference). Structure 31 depicts arepresentative structure in the presence of the metal ion and any othernecessary ligands; Structure 31 depicts uradine, although as for all thestructures herein, any other base may also be used.

[0173] L_(a) is a ligand, which may include nitrogen, oxygen, sulfur orphosphorus donating ligands or organometallic ligands such asmetallocene ligands. Suitable L_(a) ligands include, but not limited to,phenanthroline, imidazole, by and terpy. L_(r) and M are as definedabove. Again, it will be appreciated by those in the art, a conductiveoligomer may be included between the nucleoside and the electrontransfer moiety.

[0174] Similarly, as for the conductive oligomers, the linkage may bedone using a linker, which may utilize an amide linkage (see generallyTelser et al., J. Am. Chem. Soc. 111:7221-7226 (1989); Telser et al., J.Am. Chem. Soc. 111:7226-7232 (1989), both of which are expresslyincorporated by reference). These structures are generally depictedbelow in Structure 32, which again uses uridine as the base, although asabove, the other bases may also be used:

[0175] In this embodiment, L is a ligand as defined above, with L, and Mas defined above as well. Preferably, L is amino, phen, byp and terpy.

[0176] In a preferred embodiment, the second electron transfer moietyattached to a nucleoside is a metallocene; i.e. the L and L_(r) ofStructure 32 are both metallocene ligands, L_(m), as described above.Structure 33 depicts a preferred embodiment wherein the metallocene isferrocene, and the base is uridine, although other bases may be used:

[0177] Preferred metallocenes include ferrocene, cobaltocene andosmiumocene.

[0178] Thus, in a preferred embodiment, the invention providesmetallocenes covalently attached to nucleosides. In a preferredembodiment, the metallocene is attached to the base of a nucleoside. Ina preferred embodiment, the metallocene is ferrocene or substitutedferrocene.

[0179] In a preferred embodiment, the second electron transfer moiety isattached to a ribose at any position of the ribose-phosphate backbone ofthe nucleic acid, i.e. either the 5′ or 3′ terminus or any internalnucleoside. As is known in the art, nucleosides that are modified ateither the 2′ or 3′ position of the ribose can be made, with nitrogen,oxygen, sulfur and phosphorus-containing modifications possible.Amino-modified ribose is preferred. See generally PCT publication WO95/15971, incorporated herein by reference. These modification groupsmay be used as a transition metal ligand, or as a chemically functionalmoiety for attachment of other transition metal ligands andorganometallic ligands, or organic electron donor moieties as will beappreciated by those in the art. In this embodiment, a linker such asdepicted herein for “Z” may be used as well, or a conductive oligomerbetween the ribose and the electron transfer moiety. Preferredembodiments utilize attachment at the 2′ or 3′ position of the ribose,with the 2′ position being preferred. Thus for example, the conductiveoligomers depicted in Structure 13, 14 and 15 may be replaced byelectron transfer moieties; alternatively, as is depicted in Structure30, the electron transfer moieties may be added to the free terminus ofthe conductive oligomer.

[0180] In a preferred embodiment, a metallocene serves as the secondelectron transfer moiety, and is attached via an amide bond as depictedbelow in Structure 34. The examples outline the synthesis of a preferredcompound when the metallocene is ferrocene.

[0181] Amine linkages, or linkages via other heteroatoms, are alsopossible.

[0182] In a preferred embodiment, the second electron transfer moiety isattached to a phosphate at any position of the ribose-phosphate backboneof the nucleic acid. This may be done in a variety of ways. In oneembodiment, phosphodiester bond analogs such as phosphoramide orphosphoramidite linkages may be incorporated into a nucleic acid as atransition metal ligand (see PCT publication WO 95/15971, incorporatedby reference). Alternatively, the conductive oligomers depicted inStructures 16 and 17 may be replaced by electron transfer moieties;alternatively, the electron transfer moieties may be added to the freeterminus of the conductive oligomer.

[0183] Preferred electron transfer moieties for covalent attachment to asingle stranded nucleic acid include, but are not limited to, transitionmetal complexes, including metallocenes and substituted metallocenessuch as metalloceneophanes, and complexes of Ru, Os, Re and Pt.Particularly preferred are ferrocene and its derivatives (particularlyferroceneophane) and complexes of transition metals including Ru, Os, Reand Pt containing one or more amine or polyamine, imidazole,phenathroline, pyridine, bipyridine and or terpyridine and theirderivatives. For Pt, additional preferred ligands include the diiminedithiolate complexes such as quinoxaline-2,3-dithiolate complexes.

[0184] As described herein, the invention provides compositionscontaining electrodes as a first electron transfer moiety linked via aconductive oligomer to a nucleic acid which has at least a secondelectron transfer moiety covalently attached. Any combination ofpositions of electron transfer moiety attachment can be made; i.e. anelectrode at the 5′ terminus, a second electron transfer moiety at aninternal position; electrode at the 5′ terminus, second moiety at the 3′end; second moiety at the 5′ terminus, electrode at an internalposition; both electrode and second moiety at internal positions;electrode at an internal position, second moiety at the 3′ terminus,etc. A preferred embodiment utilizes both the electrode and the secondelectron transfer moiety attached to internal nucleosides.

[0185] The compositions of the invention may additionally contain one ormore labels at any position. By “label” herein is meant an element (e.g.an isotope) or chemical compound that is attached to enable thedetection of the compound. Preferred labels are radioactive isotopiclabels, and colored or fluorescent dyes. The labels may be incorporatedinto the compound at any position.

[0186] The compositions of the invention are generally synthesized asoutlined below, generally utilizing techniques well known in the art.

[0187] The compositions may be made in several ways. A preferred methodfirst synthesizes a conductive oligomer attached to the nucleoside, withaddition of additional nucleosides followed by attachment to theelectrode. A second electron transfer moiety, if present, may be addedprior to attachment to the electrode or after. Alternatively, the wholenucleic acid may be made and then the completed conductive oligomeradded, followed by attachment to the electrode. Alternatively, theconductive oligomer and monolayer (if present) are attached to theelectrode first, followed by attachment of the nucleic acid.

[0188] The latter two methods may be preferred when conductive oligomersare used which are not stable in the solvents and under the conditionsused in traditional nucleic acid synthesis.

[0189] In a preferred embodiment, the compositions of the invention aremade by first forming the conductive oligomer covalently attached to thenucleoside, followed by the addition of additional nucleosides to form anucleic acid, including, if present, a nucleoside containing a secondelectron transfer moiety, with the last step comprising the addition ofthe conductive oligomer to the electrode.

[0190] The attachment of the conductive oligomer to the nucleoside maybe done in several ways. In a preferred embodiment, all or part of theconductive oligomer is synthesized first (generally with a functionalgroup on the end for attachment to the electrode), which is thenattached to the nucleoside. Additional nucleosides are then added asrequired, with the last step generally being attachment to theelectrode. Alternatively, oligomer units are added one at a time to thenucleoside, with addition of additional nucleosides and attachment tothe electrode.

[0191] A general outline of a preferred embodiment is depicted in FIG.1, using a phenyl-acetylene oligomer as generally depicted in Structure5. Other conductive oligomers will be made using similar techniques,such as heterooligomers, or as known in the art. Thus, for example,conductive oligomers using alkene or acetylene bonds are made as isknown in the art.

[0192] The conductive oligomer is then attached to a nucleoside that maycontain one (or more) of the oligomer units, attached as depictedherein.

[0193] In a preferred embodiment, attachment is to a ribose of theribose-phosphate backbone. Thus, FIG. 1 depicts attachment via an amidelinkage, and FIG. 2 depicts the synthesis of compounds with aminelinkages.

[0194] Alternatively, attachment is via a phosphate of theribose-phosphate backbone. Examples of two synthetic schemes are shownin FIG. 4 (synthesis of Structure 16 type compounds) and FIG. 5(synthesis of Structure 16 type compounds). Although both Figures showattachment at the 3′ position of the ribose, attachment can also be madevia the 2′ position. In FIG. 5, Z is an ethylene linker, although otherlinkers may be used as well, as will be appreciated by those in the art.

[0195] In a preferred embodiment, attachment is via the base. A generalscheme is depicted in FIG. 3, using uridine as the nucleoside and aphenylene-acetylene conductive oligomer. As will be appreciated in theart, amide linkages are also possible, such as depicted in Structure 12,using techniques well known in the art.

[0196] Once the modified nucleosides are prepared, protected andactivated, prior to attachment to the electrode, they may beincorporated into a growing oligonucleotide by standard synthetictechniques (Gait, Oligonucleotide Synthesis: A Practical Approach, IRLPress, Oxford, UK 1984; Eckstein) in several ways. In one embodiment,one or more modified nucleosides are converted to the triphosphate formand incorporated into a growing oligonucleotide chain by using standardmolecular biology techniques such as with the use of the enzyme DNApolymerase I, T4 DNA polymerase, T7 DNA polymerase, Taq DNA polymerase,reverse transcriptase, and RNA polymerases. For the incorporation of a3′ modified nucleoside to a nucleic acid, terminaldeoxynucleotidyltransferase may be used. (Ratliff, Terminaldeoxynucleotidyltransferase. In The Enzymes, Vol 14A. P. D. Boyer ed. pp105-118. Academic Press, San Diego, Calif. 1981). Alternatively, andpreferably, the amino nucleoside is converted to the phosphoramidite orH-phosphonate form, which are then used in solid-phase or solutionsyntheses of oligonucleotides. In this way the modified nucleoside,either for attachment at the ribose (i.e. amino- or thiol-modifiednucleosides) or the base, is incorporated into the oligonucleotide ateither an internal position or the 5′ terminus. This is generally donein one of two ways. First, the 5′ position of the ribose is protectedwith 4′,4-dimethoxytrityl (DMT) followed by reaction with either2-cyanoethoxy-bis-diisopropylaminophosphine in the presence ofdiisopropylammonium tetrazolide, or by reaction withchlorodiisopropylamino 2′-cyanoethyoxyphosphine, to give thephosphoramidite as is known in the art; although other techniques may beused as will be appreciated by those in the art. See Gait, supra;Caruthers, Science 230:281 (1985), both of which are expresslyincorporated herein by reference.

[0197] For attachment of an electron transfer moiety to the 3′ terminus,a preferred method utilizes the attachment of the modified nucleoside tocontrolled pore glass (CPG) or other oligomeric supports. In thisembodiment, the modified nucleoside is protected at the 5′ end with DMT,and then reacted with succinic anhydride with activation. The resultingsuccinyl compound is attached to CPG or other oligomeric supports as isknown in the art. Further phosphoramidite nucleosides are added, eithermodified or not, to the 5′ end after deprotection. Thus, the presentinvention provides conductive oligomers covalently attached tonucleosides attached to solid oligomeric supports such as CPG, andphosphoramidite derivatives of the nucleosides of the invention.

[0198] The growing nucleic acid chain may also comprise at least onenucleoside with covalently attached second electron transfer moiety. Asdescribed herein, modified nucleosides with covalently attached secondelectron transfer moieties may be made, and incorporated into thenucleic acid as outlined above for the conductive oligomer-nucleosides.When a transition metal complex is used as the second electron transfermoiety, synthesis may occur in several ways. In a preferred embodiment,the ligand(s) are added to a nucleoside, followed by the transitionmetal ion, and then the nucleoside with the transition metal complexattached is added to an oligonucleotide, i.e. by addition to the nucleicacid synthesizer. Alternatively, the ligand(s) may be attached, followedby incorportation into a growing oligonucleotide chain, followed by theaddition of the metal ion.

[0199] In a preferred embodiment, electron transfer moieties areattached to a ribose of the ribose-phosphate backbone. This is generallydone as is outlined in PCT publication SO 95/15971, using amino-modifiednucleosides, at either the 2′ or 3′ position of the ribose. The aminogroup may then be used either as a ligand, for example as a transitionmetal ligand for attachment of the metal ion, or as a chemicallyfunctional group that can be used for attachment of other ligands ororganic electron transfer moieties, for example via amide linkages, aswill be appreciated by those in the art. For example, the examplesdescribe the synthesis of a nucleoside with a metallocene linked via anamide bond to the ribose.

[0200] In a preferred embodiment, electron transfer moieties areattached to a phosphate of the ribose-phosphate backbone. As outlinedherein, this may be done using phosphodiester analogs such asphosphoramidite bonds, see generally PCT publication WO 95/15971, or canbe done in a similar manner to that depicted in FIGS. 4 and 5, where theconductive oligomer is replaced by a transition metal ligand or complexor an organic electron transfer moiety.

[0201] In a preferred embodiment, electron transfer moieties areattached to a base of the nucleoside. This may be done in a variety ofways. In one embodiment, amino groups of the base, either naturallyoccurring or added as is described herein (see the figures, forexample), are used either as ligands for transition metal complexes oras a chemically functional group that can be used to add other ligands,for example via an amide linkage, or organic electron transfer moieties.This is done as will be appreciated by those in the art. Alternatively,nucleosides containing halogen atoms attached to the heterocyclic ringare commercially available. Acetylene linked ligands may be added usingthe halogenated bases, as is generally known; see for example, Tzalis etal., Tetrahedron Lett. 36(34):6017-6020 (1995); Tzalis et al.,Tetrahedron Lett. 36(2):3489-3490 (1995); and Tzalis et al., Chem.Communications (in press) 1996, all of which are hereby expresslyincorporated by reference. See also the examples, which describes thesynthesis of a metallocene attached via an acetylene linkage to thebase.

[0202] In one embodiment, the nucleosides are made with transition metalligands, incorporated into a nucleic acid, and then the transition metalion and any remaining necessary ligands are added as is known in theart. In an alternative embodiment, the transition metal ion andadditional ligands are added prior to incorporation into the nucleicacid.

[0203] In some embodiments, as outlined herein, conductive oligomers areused between the second electron transfer moieties and the nucleosides.These are made using the techniques described herein, with the additionof the terminal second electron transfer moiety.

[0204] Once the nucleic acids of the invention are made, with acovalently attached conductive oligomer and optionally a second electrontransfer moiety, the conductive oligomer is attached to the electrode.The method will vary depending on the type of electrode used. As isdescribed herein, the conductive oligomers are generally made with aterminal “A” linker to facilitate attachment to the electrode. For thepurposes of this application, a sulfur-gold attachment is considered acovalent attachment.

[0205] In a preferred embodiment, conductive oligomers are covalentlyattached via sulfur linkages to the electrode. However, surprisingly,traditional protecting groups for use of attaching molecules to goldelectrodes are generally not suitable for use in both synthesis of thecompositions described herein and inclusion in oligonucleotide syntheticreactions. Accordingly, the present invention provides novel methods forthe attachment of conductive oligomers to gold electrodes, utilizing anunusual protecting group, ethylpyridine, as is depicted in the Figures.

[0206] This may be done in several ways. In a preferred embodiment, thesubunit of the conductive oligomer which contains the sulfur atom forattachment to the electrode is protected with an ethyl-pyridine group.This is generally done by contacting the subunit containing the sulfuratom (preferably in the form of a sulfhydryl) with a vinyl pyridinegroup under conditions whereby an ethylpyridine group is added to thesulfur atom. This subunit also generally contains a functional moietyfor attachment of additional subunits, and thus additional subunits areattached to form the conductive oligomer. The conductive oligomer isthen attached to a nucleoside, and additional nucleosides attached. Theprotecting group is then removed and the sulfur-gold covalent attachmentis made. Alternatively, all or part of the conductive oligomer is made,and then either a subunit containing a protected sulfur atom is added,or a sulfur atom is added and then protected. The conductive oligomer isthen attached to a nucleoside, and additional nucleosides attached.

[0207] Alternatively, the conductive oligomer attached to a nucleic acidis made, and then either a subunit containing a protected sulfur atom isadded, or a sulfur atom is added and then protected. Alternatively, theethyl pyridine protecting group may be used as above, but removed afterone or more steps and replaced with a standard protecting group like adisulfide. Thus, the ethyl pyridine may serve as the protecting groupfor some of the synthetic reactions, and then removed and replaced witha traditional protecting group.

[0208] By “subunit” of a conductive polymer herein is meant at least themoiety of the conductive oligomer to which the sulfur atom is attached,although additional atoms may be present, including either functionalgroups which allow the addition of additional components of the,conductive oligomer, or additional components of the conductiveoligomer. Thus, for example, when Structure 2 oligomers are used, asubunit comprises at least the first Y group.

[0209] A preferred method comprises 1) adding an ethyl pyridineprotecting group to a sulfur atom attached to a first subunit of aconductive oligomer, generally done by adding a vinyl pyridine group toa sulfhydryl; 2) adding additional subunits to form said conductiveoligomer; 3) adding at least a first nucleoside to the conductiveoligomer; 4) adding additional nucleosides to the first nucleoside toform a nucleic acid; 5) attaching the conductive oligomer to the goldelectrode. This may also be done in the absence of nucleosides, as isdescribed in the Examples.

[0210] The above method may also be used to attach passavation moleculesto a gold electrode.

[0211] In a preferred embodiment, a monolayer of passavation agents isadded to the electrode. Generally, the chemistry of addition is similarto or the same as the addition of conductive oligomers to the electrode,i.e. using a sulfur atom for attachment to a gold electrode, etc.Compositions comprising monolayers in addition to the conductiveoligomers covalently attached to nucleic acids (with or without secondelectron transfer moieties) may be made in at least one of five ways:(1) addition of the monolayer, followed by subsequent addition of theconductive oligomer-nucleic acid complex; (2) addition of the conductiveoligomer-nucleic acid complex followed by addition of the monolayer; (3)simultaneous addition of the monolayer and conductive oligomer-nucleicacid complex; (4) formation of a monolayer (using any of 1, 2 or 3)which includes conductive oligomers which terminate in a functionalmoiety suitable for attachment of a completed nucleic acid; or (5)formation of a monolayer which includes conductive oligomers whichterminate in a functional moiety suitable for nucleic acid synthesis,i.e. the nucleic acid is synthesized on the surface of the monolayer asis known in the art. Such suitable functional moieties include, but arenot limited to, nucleosides, amino groups, carboxyl groups, protectedsulfur moieties, or hydroxyl groups for phosphoramidite additions. Theexamples describe the formation of a monolayer on a gold electrode usingthe preferred method (1).

[0212] As will be appreciated by those in the art, electrodes may bemade that have any combination of nucleic acids, conductive oligomersand passavation agents. Thus, a variety of different conductiveoligomers or passavation agents may be used on a single electrode.

[0213] Once made, the compositions find use in a number of applications,as described herein.

[0214] In a preferred embodiment, the compositions of the invention areused as probes in hybridization assays to detect target sequences in asample. The term “target sequence” or grammatical equivalents hereinmeans a nucleic acid sequence on a single strand of nucleic acid. Thetarget sequence may be a portion of a gene, a regulatory sequence,genomic DNA, cDNA, RNA including mRNA and rRNA, or others. It may be anylength, with the understanding that longer sequences are more specific.As will be appreciated by those in the art, the complementary targetsequence may take many forms. For example, it may be contained within alarger nucleic acid sequence, i.e. all or part of a gene or mRNA, arestriction fragment of a plasmid or genomic DNA, among others. As isoutlined more fully below, probes are made to hybridize to targetsequences to determine the presence or absence of the target sequence ina sample. Generally speaking, this term will be understood by thoseskilled in the art.

[0215] The probes of the present invention are designed to becomplementary to the target sequence, such that hybridization of thetarget sequence and the probes of the present invention occurs. Asoutlined below, this complementarity need not be perfect; there may beany number of base pair mismatches which will interfere withhybridization between the target sequence and the single strandednucleic acids of the present invention. However, if the number ofmutations is so great that no hybridization can occur under even theleast stringent of hybridization conditions, the sequence is not acomplementary target sequence.

[0216] A variety of hybridization conditions may be used in the presentinvention, including high, moderate and low stringency conditions; seefor example Maniatis et al., Molecular Cloning: A Laboratory Manual, 2dEdition, 1989, and Short Protocols in Molecular Biology, ed. Ausubel, etal, hereby incorporated by referenece. The hybridization conditions mayalso vary when a non-ionic backbone, i.e. PNA is used, as is known inthe art.

[0217] In a preferred embodiment, single stranded nucleic acids are madewhich contain a first electron transfer moiety, an electrode, and atleast a second electron tranfer moiety. Hybridization to a targetsequence forms a double stranded hybridization complex. In ahybridization complex, at least the sequence between the nucleosidescontaining the electron transfer moieties is double stranded, i.e.contains stacked π-orbitals, such that upon initiation, the complex iscapable of transferring at least one electron from one of the electrontransfer moieties to the other. As will be appreciated by those in theart, an electrode may serve as either an electron donor or acceptor, andthe choice of the second electron transfer species is made accordingly.

[0218] In an alternative embodiment, compositions comprising a) a firstsingle stranded nucleic acid covalently attached to an electrode via aconductive oligomer and b) a second single stranded nucleic acidcontaining a second electron transfer moiety, are made. In thisembodiment, the first single stranded nucleic acid is capable ofhybridizing to a first target domain, and the second single strandednucleic acid is capable of hybridizing to a second target domain. Theterms “first target domain” and “second target domain” or grammaticalequivalents herein means two portions of a target sequence within anucleic acid which is under examination. The first target domain may bedirectly adjacent to the second target domain, or the first and secondtarget domains may be separated by an intervening target domain.Preferably, there are no gaps between the domains; i.e. they arecontiguous. The terms “first” and “second” are not meant to confer anorientation of the sequences with respect to the 5′-3′ orientation ofthe target sequence. For example, assuming a 5′-3′ orientation of thecomplementary target sequence, the first target domain may be locatedeither 5′ to the second domain, or 3′ to the second domain.

[0219] In this embodiment, the first single stranded nucleic acid ishybridized to the first target domain, and the second single strandednucleic acid is hybridized to the second target domain to form ahybridization complex. As outlined above, the hybridization complex isthen capable of transferring at least one electron between the electrontransfer moieties upon initiation.

[0220] In one embodiment, compositions comprising a) a single strandednucleic acid covalently attached to an electrode via a conductiveoligomer, and b) a target nucleic acid are made. In this embodiment,once hybridization of the target and the probe occurs, an electrontransfer moiety that will preferentially associate with double strandednucleic acid is added, similar to the method of Millan et al., Anal.Chem. 65:2317-2323 (1993); Millan et al., Anal. Chem. 662943-2948(1994), both of which are hereby expressly incorporated by reference.For example, intercalators may be used; since intercalation generallyonly occurs in the presence of double stranded nucleic acid, only in thepresence of target hybridization will electron transfer occur.Intercalating transition metal complex electron transfer moieties areknown in the art.

[0221] A further embodiment utilizes compositions comprising a) a firstsingle stranded nucleic acid covalently attached to an electrode via aconductive oligomer; b) a second single stranded nucleic acid containinga second electron transfer moiety; and c) an intervening single strandednucleic acid, which may or may not be labelled or contain an electrontransfer moiety. As generally outlined in PCT WO 95/15971, the firstsingle stranded nucleic acid hybridizes to the first target domain, thesecond single stranded nucleic acid hybridizes to the second targetdomain, and the intervening nucleic acid hybridizes to the interveningtarget domain, with electron transfer upon initiation. The interveningnucleic acid may be any length, taking into consideration the parametersfor the distance between the electron transfer moieties. In addition,intervening sequences of greater than about 10 to 15 nucleosides aredesirable, since it is more likely to remain hybridized to form a doublestranded nucleic acid, although it may be a single nucleoside.

[0222] In addition, the first and second, or first, intervening andsecond, nucleic acids may be ligated together prior to the electrontransfer reaction, using standard molecular biology techniques such asthe use of a ligase.

[0223] In one embodiment, the compositions of the invention are used todetect mutations in a complementary target sequence. A mutation, whetherit be a substitution, insertion or deletion of a nucleoside ornucleosides, results in incorrect base pairing in a hybridized doublehelix of nucleic acid. Accordingly, if the path of an electron from anelectron donor moiety to an electron acceptor moiety spans the regionwhere the mismatch lies, the electron transfer will be reduced such thata change in the relative impediance will be seen. Therefore, in thisembodiment, the electron donor moiety is attached to the nucleic acid ata 5′ position from the mutation, and the electron acceptor moiety isattached at a 3′ position, or vice versa.

[0224] Electron transfer is generally initiated electronically, withvoltage being preferred. A potential is applied to a sample containingmodified nucleic acid probes. Precise control and variations in theapplied potential can be via a potentiostat and either a three electrodesystem (one reference, one sample and one counter electrode) or a twoelectrode system (one sample and one counter electrode). This allowsmatching of applied potential to peak electron transfer potential of thesystem which depends in part on the choice of electron acceptorsattached to the nucleic acid and in part on the conductive oligomerused. As described herein, ferrocene is a preferred electron transfermoiety.

[0225] Preferably, initiation and detection is chosen to maximize therelative difference between the impediences of double stranded nucleicacid and single stranded nucleic acid. The efficiency of electrontransfer through nucleic acid is a function of the impedience of thecompound.

[0226] In a preferred embodiment, an input electron source in solutionis used in the initiation of electron transfer, when a passavation agentmonolayer is present on the electrode. This may be done in two generalways. In a preferred embodiment, an input electron source is used thathas a lower redox potential than the second electron transfer moiety(ETM) covalently attached to the probe nucleic acid. For example,ferrocene, as a second ETM attached to the compositions of the inventionas described in the examples, has a redox potential of roughly 420 mV.Ferrocyanide, an electron source, has a redox potential of roughly 170mV. At voltages less than the redox potential of the ETM, but higherthan the redox potential of the electron source, i.e. 170-420 mV,ferrocyanide is oxided, but is unable to donate an electron to ferrocenein the +2 state; i.e. no electron transfer occurs. However, at or abovethe redox potential of ferrocene, ferrocene is converted to ferricenium,which then transfers an electron to the nucleic acid. If this nucleicacid is double stranded, transfer proceeds rapidly through the doublestranded nucleic acid, through the conductive oligomer, to theelectrode. Now the oxidized ferricyanide in solution can transfer anelectron to the ETM. In this way, the electron source (or co-reductant)serves to amplify the signal generated in the system, as the electronsource molecules rapidly and repeatedly donate electrons to the secondETM attached to the nucleic acid. The use of electron source molecules,however, is only possible when an insulating or passavation layer ispresent, since otherwise the source molecule will transfer electronsdirectly to the electrode. Accordingly, in a preferred embodiment, anelectron source is used in solution to amplify the signal generated inthe presence of hybridized target sequence.

[0227] In an alternate preferred embodiment, an input electron source isused that has a higher redox potential than the second electron transfermoiety (ETM) covalently attached to the probe nucleic acid. For example,luminol, an electron source, has a redox potential of roughly 720 mV. Atvoltages higher than the redox potential of the ETM, but lower than theredox potential of the electron source, i.e. 420-720 mV, the ferroceneis oxided, and transfers a single electron to the electrode via theconductive oligomer. However, the ETM is unable to accept any electronsfrom the luminol electron source, since the voltages are less than theredox potential of the luminol. However, at or above the redox potentialof luminol, the luminol then transfers an electron to the ETM, allowingrapid and repeated electron transfer. In this way, the electron source(or co-reductant) serves to amplify the signal generated in the system,as the electron source molecules rapidly and repeatedly donate electronsto the second ETM attached to the nucleic acid.

[0228] Luminol has the added benefit of becoming a chemiluminiscentspecies upon oxidation, thus allowing photo-detection of electrontransfer through double-stranded nucleic acid. Thus, as long as theluminol is unable to contact the electrode directly, i.e. in thepresence of a passavation layer, luminol can only be oxidized bytransferring an electron to the second electron transfer moiety on thenucleic acid (e.g. ferrocene). When double stranded nucleic acid is notpresent, i.e. when the target sequence is not hybridized to thecomposition of the invention, each second electron transfer moiety has ahigh impediance, resulting in a low photon emission and thus a low (ifany) signal from the luminol. In the presence of double stranded nucleicacid, i.e. target sequence hybridization, the second electron transfermoieties have low impediance, thus generating a much larger signal.Thus, the measure of luminol oxidation by photon emission is an indirectmeasurement of the ability of the second electron transfer moiety todonate electrons to the electrode. Furthermore, since photon detectionis generally more sensitive than electronic detection, the sensitivityof the system is increased.

[0229] Suitable electron source molecules are well known in the art, andinclude, but are not limited to, ferricyanide, and luminol.

[0230] Alternatively, output electron acceptors or sinks could be used,i.e. the above reactions could be run in reverse, with the ETM such as ametallocene receiving an electron from the electrode, converting it tothe metallicenium, with the output electron acceptor then accepting theelectron rapidly and repeatedly. In this embodiment, cobalticenium isthe preferred ETM.

[0231] Electron transfer through nucleic acid can be detected in avariety of ways. A variety of detection methods may be used, including,but not limited to, optical detection, which includes fluorescence,phosphorescence, luminiscence, chemiluminescence,electrochemiluminescence, and refractive index; and electronicdetection, including, but not limited to, amperommetry, voltammetry,capacitance and impedence. These methods include time or frequencydependent methods based on AC or DC currents, pulsed methods, lock-intechniques, filtering (high pass, low pass, band pass), andtime-resolved techniques including time-resolved fluoroscence. In someembodiments, all that is required is electron transfer detection; inothers, the rate of electron transfer may be determined.

[0232] In one embodiment, the efficient transfer of electrons from oneend of a nucleic acid double helix to the other results in stereotypedchanges in the redox state of both the electron donor and acceptor. Withmany electron transfer moieties including the complexes of rutheniumcontaining bipyridine, pyridine and imidazole rings, these changes inredox state are associated with changes in spectral properties.Significant differences in absorbance are observed between reduced andoxidized states for these molecules. See for example Fabbrizzi et al.,Chem. Soc. Rev. 1995 pp197-202). These differences can be monitoredusing a spectrophotometer or simple photomultiplier tube device.

[0233] In this embodiment, possible electron donors and acceptorsinclude all the derivatives listed above for photoactivation orinitiation. Preferred electron donors and acceptors havecharacteristically large spectral changes upon oxidation and reductionresulting in highly sensitive monitoring of electron transfer. Suchexamples include Ru(NH₃)₄py and Ru(bpy)₂im as preferred examples. Itshould be understood that only the donor or acceptor that is beingmonitored by absorbance need have ideal spectral characteristics. Thatis, the electron acceptor can be optically invisible if only theelectron donor is monitored for absorbance changes.

[0234] In a preferred embodiment, the electron transfer is detectedfluorometrically. Numerous transition metal complexes, including thoseof ruthenium, have distinct fluorescence properties. Therefore, thechange in redox state of the electron donors and electron acceptorsattached to the nucleic acid can be monitored very sensitively usingfluorescence, for example with Ru(4,7-biphenyl₂-phenanthroline)₃ ²⁺. Theproduction of this compound can be easily measured using standardfluorescence assay techniques. For example, laser induced fluorescencecan be recorded in a standard single cell fluorimeter, a flow through“on-line” fluorimeter (such as those attached to a chromatographysystem) or a multi-sample “plate-reader” similar to those marketed for96-well immuno assays.

[0235] Alternatively, fluorescence can be measured using fiber opticsensors with nucleic acid probes in solution or attached to the fiberoptic. Fluorescence is monitored using a photomultiplier tube or otherlight detection instrument attached to the fiber optic. The advantage ofthis system is the extremely small volumes of sample that can beassayed.

[0236] In addition, scanning fluorescence detectors such as theFluorImager sold by Molecular Dynamics are ideally suited to monitoringthe fluorescence of modified nucleic acid molecules arrayed on solidsurfaces. The advantage of this system is the large number of electrontransfer probes that can be scanned at once using chips covered withthousands of distinct nucleic acid probes.

[0237] Many transition metal complexes display fluorescence with largeStokes shifts. Suitable examples include bis- and trisphenanthrolinecomplexes and bis- and trisbipyridyl complexes of transition metals suchas ruthenium (see Juris, A., Balzani, V., et. al. Coord. Chem. Rev., V.84, p. 85-277, 1988). Preferred examples display efficient fluorescence(reasonably high quantum yields) as well as low reorganization energies.These include Ru(4,7-biphenyl₂-phenanthroline)₃ ²⁺,Ru(4,4′-diphenyl-2,2′-bipyridine)₃ ²⁺ and platinum complexes (seeCummings et al., J. Am. Chem. Soc. 118:1949-1960 (1996), incorporated byreference).

[0238] Alternatively, a reduction in fluorescence associated withhybridization can be measured using these systems. An electron transfer“donor” molecule that fluoresces readily when on single stranded nucleicacid (with an “acceptor” on the other end) will undergo a reduction influorescent intensity when complementary nucleic acid binds the probeallowing efficient transfer of the excited state electron. This drop influorescence can be easily monitored as an indicator of the presence ofa target sequence using the same methods as those above.

[0239] In a further embodiment, electrochemiluminescence is used as thebasis of the electron transfer detection. With some electron transfermoieties such as Ru²⁺(bpy)₃, direct luminescence accompanies excitedstate decay. Changes in this property are associated with nucleic acidhybridization and can be monitored with a simple photomultiplier tubearrangement (see Blackburn, G. F. Clin. Chem. 37: 1534-1539 (1991); andJuris et al., supra.

[0240] In a preferred embodiment, electronic detection is used,including amperommetry, voltammetry, capacitance, and impedence.Suitable techniques include, but are not limited to, electrogravimetry;coulometry (including controlled potential coulometry and constantcurrent coulometry); voltametry (cyclic voltametry, pulse voltametry(normal pulse voltametry, square wave voltametry, differential pulsevoltametry, Osteryoung square wave voltametry, and coulostatic pulsetechniques); stripping analysis (aniodic stripping analysis, cathiodicstripping analysis, square wave stripping voltammetry); conductancemeasurements (electrolytic conductance, direct analysis); time-dependentelectrochemical analyses (chronoamperometry, chronopotentiometry, cyclicchronopotentiometry and amperometry, AC polography, chronogalvametry,and chronocoulometry); AC impedance measurement; capacitancemeasurement; and photoelectrochemistry.

[0241] In a preferred embodiment, monitoring electron transfer throughnucleic acid is via amperometric detection. This method of detectioninvolves applying a potential (as compared to a separate referenceelectrode) between the nucleic acid-conjugated electrode and anauxiliary (counter) electrode in the sample containing target genes ofinterest. Electron transfer of differing efficiencies is induced insamples in the presence or absence of target nucleic acid; that is, thepresence or absence of the target nucleic acid alters the impedience ofthe nucleic acid (i.e. double stranded versus single stranded) which canresult in different currents.

[0242] The device for measuring electron transfer amperometricallyinvolves sensitive current detection and includes a means of controllingthe voltage potential, usually a potentiostat. This voltage is optimizedwith reference to the potential of the electron donating complex on thenucleic acid. Possible electron donating complexes include thosepreviously mentioned with complexes of iron, osmium, platinum, cobalt,rhenium and ruthenium being preferred and complexes of iron being mostpreferred.

[0243] In a preferred embodiment, alternative electron detection modesare utilized.

[0244] For example, potentiometric (or voltammetric) measurementsinvolve non-faradaic (no net current flow) processes and are utilizedtraditionally in pH and other ion detectors. Similar sensors are used tomonitor electron transfer through nucleic acid. In addition, otherproperties of insulators (such as resistance) and of conductors (such asconductivity, impedance and capicitance) could be used to monitorelectron transfer through nucleic acid. Finally, any system thatgenerates a current (such as electron transfer) also generates a smallmagnetic field, which may be monitored in some embodiments.

[0245] It should be understood that one benefit of the fast rates ofelectron transfer observed in the compositions of the invention is thattime resolution can greatly enhance the signal-to-noise results ofmonitors based on absorbance, fluorescence and electronic current. Thefast rates of electron transfer of the present invention result both inhigh signals and stereotyped delays between electron transfer initiationand completion. By amplifying signals of particular delays, such asthrough the use of pulsed initiation of electron transfer and “lock-in”amplifiers of detection, between two and four orders of magnitudeimprovements in signal-to-noise may be achieved.

[0246] Thus, the compositions of the present invention may be used in avariety of research, clinical, quality control, or field testingsettings.

[0247] In a preferred embodiment, the probes are used in geneticdiagnosis. For example, probes can be made using the techniquesdisclosed herein to detect target sequences such as the gene fornonpolyposis colon cancer, the BRCA1 breast cancer gene, P53, which is agene associated with a variety of cancers, the Apo E4 gene thatindicates a greater risk of Alzheimer's disease, allowing for easypresymptomatic screening of patients, mutations in the cystic fibrosisgene, or any of the others well known in the art.

[0248] In an additional embodiment, viral and bacterial detection isdone using the complexes of the invention. In this embodiment, probesare designed to detect target sequences from a variety of bacteria andviruses. For example, current blood-screening techniques rely on thedetection of anti-HIV antibodies. The methods disclosed herein allow fordirect screening of clinical samples to detect HIV nucleic acidsequences, particularly highly conserved HIV sequences. In addition,this allows direct monitoring of circulating virus within a patient asan improved method of assessing the efficacy of anti-viral therapies.Similarly, viruses associated with leukemia, HTLV-I and HTLV-II, may bedetected in this way. Bacterial infections such as tuberculosis may alsobe detected.

[0249] In a preferred embodiment, the nucleic acids of the inventionfind use as probes for toxic bacteria in the screening of water and foodsamples. For example, samples may be treated to lyse the bacteria torelease its nucleic acid, and then probes designed to recognizebacterial strains, including, but not limited to, such pathogenicstrains as, Salmonella, Campylobacter, Vibrio cholerae, enterotoxicstrains of E. coli, and Legionnaire's disease bacteria. Similarly,bioremediation strategies may be evaluated using the compositions of theinvention.

[0250] In a further embodiment, the probes are used for forensic “DNAfingerprinting” to match crime-scene DNA against samples taken fromvictims and suspects.

[0251] In an additional embodiment, the probes in an array are used forsequencing by hybridization.

[0252] The present invention also finds use as a unique methodology forthe detection of mutations in target nucleic acid sequences. As aresult, if a single stranded nucleic acid containing electron transfermoieties is hybridized to a target sequence with a mutation, theresulting perturbation of the base pairing of the nucleosides willmeasurably affect the electron transfer rate. This is the case if themutation is a substitution, insertion or deletion. Alternatively, twosingle stranded nucleic acids each with a covalently attached electrontransfer species that hybridize adjacently to a target sequence may beused. Accordingly, the present invention provides for the detection ofmutations in target sequences.

[0253] Thus, the present invention provides for extremely specific andsensitive probes, which may, in some embodiments, detect targetsequences without removal of unhybridized probe. This will be useful inthe generation of automated gene probe assays.

[0254] In an alternate embodiment the electron transfer moieties are onseparate strands. In this embodiment, one single stranded nucleic acidhas an electrode covalently attached via a conductive oligomer. Theputative target sequences are labelled with a second electron transfermoiety as is generally described herein, i.e. by incorporating anelectron transfer moiety to individual nucleosides of a PCR reactionpool. Upon hybridization of the two single-stranded nucleic acids,electron transfer is detected.

[0255] Alternatively, the compositions of the invention are useful todetect successful gene amplification in PCR, thus allowing successfulPCR reactions to be an indication of the presence or absence of a targetsequence. PCR may be used in this manner in several ways. For example,in one embodiment, the PCR reaction is done as is known in the art, andthen added to a composition of the invention comprising the targetnucleic acid with a second ETM, covalently attached to an electrode viaa conductive oligomer with subsequent detection of the target sequence.Alternatively, PCR is done using nucleotides labelled with a second ETM,either in the presence of, or with subsequent addition to, an electrodewith a conductive oligomer and a target nucleic acid. Binding of the PCRproduct containing second ETMs to the electrode composition will allowdetection via electron transfer. Finally, the nucleic acid attached tothe electrode via a conductive polymer may be one PCR primer, withaddition of a second primer labelled with an ETM. Elongation results indouble stranded nucleic acid with a second ETM and electrode covalentlyattached. In this way, the present invention is used for PCR detectionof target sequences.

[0256] In an additional embodiment, the present invention provides novelcompositions comprising metallocenes covalently attached via conductiveoligomers to an electrode, such as are generally depicted in Structure35:

[0257] Structure 35 utilizes a Structure 4 conductive oligomer, althoughas will be appreciated by those in the art, other conductive oligomerssuch as Structures 2, 3, 9 or 10 types may be used. Preferredembodiments of Structure 35 are depicted below.

[0258] Preferred R groups of Structure 37 are hydrogen.

[0259] These compositions are synthesized as follows. The conductiveoligomer linked to the metallocene is made as described herein; seealso, Hsung et al., Organometallics 14:4808-4815 (1995); and Bumm etal., Science 271:1705 (1996), both of which are expressly incorporatedherein by reference. The conductive oligomer is then attached to theelectrode using the novel ethylpyridine protecting group, as outlinedherein.

[0260] Once made, these compositions have unique utility in a number ofapplications, including photovoltaics, and infrared detection. Apreferred embodiment utilizes these compounds in calibrating apotentiostat, serving as an internal electrochemistry reference in anarray of the invention.

[0261] The following examples serve to more fully describe the manner ofusing the above-described invention, as well as to set forth the bestmodes contemplated for carrying out various aspects of the invention. Itis understood that these examples in no way serve to limit the truescope of this invention, but rather are presented for illustrativepurposes. All references cited herein are incorporated by reference.

EXAMPLES Example 1 Synthesis of Conductive Oligomer Linked via an Amideto a Nucleoside

[0262] This synthesis is depicted in FIG. 1, using uridine as thenucleoside and a Structure 4 phenyl-acetylene conductive oligomer.

[0263] Compound #1: To a solution of 10.0 gm (40 mmol) of4-iodothioanisole in 350 mL of dichloromethane cooled in an ice-waterbath was added 10.1 gm of mCPBA. The reaction mixture was stirred forhalf hour and the suspension was formed. To the suspension was added 4.0gm of powered Ca(OH)₂, the mixture was stirred at room temperature for15 min and filtered off and the solid was washed once with 30 mL ofdichloromethane. To the combined filtrate was added 12 mL oftrifluoroacetic anhydride and the reaction mixture was refluxed for 1.5h under Argon. After removing the solvents, the residue was dissolved in200 mL of a mixture of TEA and methanol (ratio=50:50) and concentratedto dryness. The residue was dissolved in 100 mL of dichloromethane andthe solution was washed once with 60 mL of the saturated ammonimchloride solution. The aqueous layer was extracted twice withdichloromethane (2×70 mL). The organic extracts were combined and driedover anhydrous sodium sulfate and immediately concentrated to dryness asquickly as possible. The residue was dissolved in 120 mL of benzene,followed by adding 5.3 mL of 4-vinylpyridine. The reaction mixture wasrefluxed under Argon overnight. The solvent was removed and the residuewas dissolved in dichloromethane for column chromatography. Silica gel(150 gm) was packed with 20% ethyl acetate/hexane mixture. The crudeproduct solution was loaded and the column was eluted with 20 to 60%ethyl acetate/hexane mixture. The fractions was identified by TLC(EtOAc: Hexane=50:50, Rf=0.24) and pooled and concentrated to dryness toafford 7.4 gm (54.2%) of the solid title compound.

[0264] Compound #2: To a solution of 3.4 gm (9.97 mmol) of Compound #1in 70 mL of diethylamine was added 200 mg ofbis(triphenylphosphine)palladium (II) chloride, 100 mg of cuprous iodideand 1.9 mL of trimethylsilylacetylene under Argon. The reaction mixturewas stirred for 2 h. After removing the diethylamine, the residue wasdissolved in dichloromethane for column chromatography. Silica gel (120gm) was packed with a cosolvent of 50% ethyl acetate/50% hexane. Thecrude sample solution was loaded and the column was eluted with the samecosolvent. After removing the solvents, the liquid title compound (2.6gm, 83.7%) was obtained.

[0265] Compound #3: To a solution of 2.6 gm of Compound #2 in 150 mL ofdichloromethane colled in an ice-water bath was added 9.0 mL of 1 Ntetrabutylammonium fluoride THF solution. The reaction mixture wasstirred for 1 h. and washed once with water and dried over anhydrousNa₂SO₄. After removing the solvent, the residue was used for columnseparation. Silica gel (50 gm) was packed with a coslovent of 50% ethylacetate/50% hexane. The crude product solution was loaded and the columnwas eluted with the same solvents. The removal of the solvents gave thesolid title compound (1.87 gm, 94.1%).

[0266] Compound #4: To a glass bottle were added 1.80 gm (7.52 mmol) ofCompound #3, 160 mg of bis(triphenylphosphine)palladium (II) chloride,80 mg of cuprous iodide and 2.70 gm (9.0 mmol) of1-trimethylsilyl-2-(4-iodophenyl)acetylene. The bottle was sealed andbubbled with Argon. Diethylamine was introduced by a syringe. Thereaction mixture was heated at 50° C. under Argon for 1 h. The amine wasremoved and the residue was dissolved in dichloromethane for theseparation. Silica gel (100 gm) was packed with 60% ethylacetate/hexane. The crude mixture was loaded and the column was elutedwith the same solvents. The fractions were identified by TLC (EtOAc:Hexane=50:50, the product emitted blue light) and pooled. The removal ofthe solvents gave the solid title product (2.47 gm, 79.8%).

[0267] Compound #5: To a solution of 2.47 gm of Compound #4 in 130 mL ofdichloromethane cooled in an ice-water bath was added 8.0 mL of 1 Ntetrabutylammonium fluoride THF solution. The reaction mixture wasstirred for 1 h. and washed once with water and dried over anhydrousNa₂SO₄. After removing the solvent, the residue was used for columnseparation. Silica gel (60 gm) was packed with a coslovent of 50% ethylacetate/50% CH₂Cl₂. The crude solution was loaded and the column waseluted with the same solvents. The removal of solvents gave the solidtitle product (1.95 gm, 95.7%).

[0268] Compound #6: To a glass bottle were added 0.23 gm (0.68 mmol) ofCompound #5, 0.5 gm (0.64 mmol) of 2′-deoxy-2′-(4-iodophenylcarbonyl)amino-5′-O-DMT uridine, 60 mg of bis(triphenylphosphine)palladium (II)chloride, 30 mg of cuprous iodide. The bottle was sealed and bubbledwith Argon. Pyrrodine(15 mL) and DMF(15 mL) were introduced by asyringe. The reaction mixture was heated at 85° C. overnight. Thesolvents were removed in vacuo and the residue was dissolved in 300 mLof dichloromethane. The solution was washed three times with water anddried over sodium sulfate. After removing the solvent, the residue wassubjected to column purification. Silica gel (30 gm) was packed with 1%TEA/1% methanol/CH2Cl2 and the sample solution was loaded. The columnwas eluted with 1% TEA/1% methanol/CH₂Cl₂ and 1% TEA/2% methanol/CH₂Cl₂.The fractions were identified and concentrated to dryness. The separatedproduct was subjected to another reverse-phase column purification.Reverse-phase silica gel(C-18, 120 gm) was packed with 60% CH₃CN/40% H₂Oand the sample was dissolved in very small amount of THF and loaded. Thecolumn was eluted with 100 mL of 60% CH₃CN/40% H₂O, 100 mL of 70%CH₃CN/30% H₂O, 100 mL of 60% CH₃CN/10% THF/30% H₂O, 200 mL of 50%CH₃CN/20% THF/30% H₂O and 500 mL of 35% CH₃CN/35% THF/30% H₂O. Thefractions were identified by HPLC (0.1 mM TEAA: CH3CN=20:80, flowrate=1.0 mL/min). and concentrated to dryness to afford a pure titlecompound.

[0269] Compound #7: To a solution of 100 mg(0.1 mmol) of pure compound#6 in 40 mL of pyridine were added 50 mgm of DMAP and 1.0 gm (10 mmol)of succinic anhydride. The reaction mixture was stirred under Argon for40 h. After removing pyridine, the residue was dissolved in 300 mL ofdichloromethane, followed by adding 150 mL of 5% aqueous NaHCO₃solution. The mixture was vigorously stirred for 3 h and separated. Theorganic layer was washed once with 1% citric acid solution and driedover anhydrous sodium sulfate and concentrated to dryness to give 110mgm of Compound #7. Without further purification, the Compound #7 wasused for the preparation of the corresponding CPG.

[0270] Conductive oligomer-Uridine-CPG: To 1.4 gm of LCAA-CPG(500 _) in100 mL round bottom flask were added 110 mgm(101 μmol) of the Compound#7, 100 mgm (230 μmol) of BOP reagent, 30 mgm (220 μmol) of HBT, 70 mLof dichloromethane and 2 mL of TEA. The mixture was shaken for threedays. The CPG was filtered off and washed twice with dichloromethane andtransferred into another 100 mL flask. Into CPG were added 50 mL ofpyridine, 10 mL of acetic anhydride and 2 mL of N-methylimidizole. TheCPG was filtered off, washed twice with pyridine, methanol,dichloromethane and ether, and dried over a vacuum. The loading of thenucleoside was measured according to the standard procedure to be 7.1μmol/gm.

[0271] 2′-Deoxy-2′-(4-iodophenylcarbonyl)amino-5′-O-DMT uridine: To asolution of 5.1 gm(9.35 mmol) of 2′-deoxy-2′-amino-5′-O-DMT uridine in250 mL of pyridine cooled in an ice-water bath was added 3 mL ofchlorotrimethylsilane. The reaction mixture was warmed up to roomtemperature and stirred for 1 h. To the prepared solution were added 0.1gm of DMAP and 3.0 gm (10.9 mmol) 4-iodobezoyl chloride and the reactionmixture was stirred overnight. To this solution was added 30 mL ofconcentrated ammonium hydroxide solution and the mixure was stirred forexact 15 min. The solvents were removed in vacuo. The residue wasdissolved in 15 mL of dichloromethane for column separation. Silica gel(125 gm) was packed with 1% TEA/2% CH₃OH/CH₂Cl₂. After loading thesample, the column was eluted with 300 mL of 1% TEA/2% CH₃OH/CH₂Cl₂, and500 mL of 1% TEA/4% CH₃OH/CH₂Cl₂. The fractions were identified by TLC(CH₃OH: CH₂Cl₂=10:90) and pooled and concentrated to dryness to give 6.2gm (85.5%) of the pure title compound.

[0272] Synthesis of the Phosphormidite (Compound #8).

[0273] To a solution of 0.2 gm of Compound #6 and 30 mg ofdiisopropylammonium tetrazolide in 10 mL of dry dichloromethane is added0.12 gm of 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphane under Argon.The solution was stirred for 5 h and diluted by adding 60 mL ofdichloromethane. The solution was washed twice with 2.5% w/v sodiumbicarbonate solution, once with the brine and dried over sodium sulfate.After removing the solvent, residue was dissolved in 5 mL ofdichloromethane, followed by adding slowly 100 mL of hexane. Thesuspension was stored at −20° C. for 1 h. The supernatant was decantedand the residue was dried over a high vacuum overnight to afford 0.19 gm(79.0%) of the title product, which will be used for DNA synthesis.

Example 2 Synthesis of a Conductive Oligomer Linked to the Ribose of aNucleoside via an Amine Linkage

[0274] Synthesis of 2′-(4-iodophenyl)amino-2′-deoxy-5′-O-DMT-uridine(Product 4): This synthesis is depicted in FIG. 2, and reference is madeto the labelling of the products on the figure. To a solution of 5.0 gmof 5′-O-DMT-uridine (Product 1) and 2.7 gm of dimethylaminopyridine in200 mL of acetonitrile was added 3.3 gm of p-iodophenyl isocyalidedichloride portion by portion under Argon. The reaction mixture wasstirred overnight. The mixture was diluted by adding 550 mL ofdichloromethane and washed twice with 5% sodium bicarbonate aqueoussolution and once with the brine solution, and then dried over sodiumsulfate. The removal of the solvent in vacuo gave the crude Product 2.Without further purification, Product 2 was dissolved in 50 mL of dryDMF and the solution was heated at 150° C. foe 2 h. After distillationof DMF, the residue was dissolved in 300 mL of dichloromethane, washedonce with 5% sodium bicarbonate solution, once with the brine solutionand dried over sodium sulfate. The removal of the solvent gave the crudeProduct 3. Without purification, the Product 3 was dissolved 100 mL of amixture of 50% Dioxane and 50% Methanol. To this solution was added 43mL of 1N NaOH solution. The reaction mixture was stirred overnight. Themixture was diluted by adding 800 mL of dichloromethane and washed twicewater and dried over Na₂SO₄. After removing the solvent, the residue wasdissolved in 15 mL of dichloromethane for the column separation. Silicagel (100 gm) of packed with 1% TEA/2% Ethanol/CH₂Cl₂, after loading thesample solution, the column was eluted with 1% TEA/2-3% Ethanol/CH₂Cl₂.The fractions were identified by TLC (CH3 OH: CH2Cl2=1:9) and pooled andconcentrated to give 2.0 gm (29.2%) of the Product 4.

[0275] Additional conductive oligomer units can then be added to product4 as outlined herein, with additional nucleotides added and attachmentto an electrode surface as described herein.

Example 3 Synthesis of a Conductive Oligomer with an R Group Attached tothe Y Aromatic Group

[0276] This synthesis is depicted in FIG. 6.

[0277] Synthesis of 2-Acetyl-5-iodotoluene (P1). To a suspension of 20gm of aluminum trichloride in 500 mL of dichloromethane was added 10.2mL of acetyl chloride under Argon. After the reaction mixture wasstirred for 15 min, 3-iodotoluene (20 gm) was added through a syringe.The mixture was stirred overnight under Argon and poured into 500 gm ofice-water. Organic layer was separated and washed once with thesaturated ammonium chloride solution, and washed once with 10% sodiumthiosulfate solution and dried over sodium sulfate. After removing thesolvent, the residue was dissolved in hexane for the columnpurification. Silica gel (260 gm) was packed with hexane, after loadingthe sample solution, the column was eluted with 750 mL of hexane, 750 mLof 1% v/v ether/hexane, 750 mL of 2% v/v ether/hexane and 1500 mL of 3%v/v ether/hexane. The fractions containing the right isomer wereidentified by GC-MS and ¹H NMR and pooled and concentrated to dryness toafford 12.2 gm (51.2%) of the title product (P 1).

[0278] Iodo-3-methyl-4-(ehynyl trimethylsilyl) benzene (P2). Under inertatmosphere 500 ml bound bottom flask was charged with 25 ml of dry THF,cooled to −78° C. and 14 ml of 2.0 M LDA solution(heptane/ethylbenzene/THF solution) was added by syringe. To thissolution 6.34 gr (24.38 mmole) of iodo-3-methyl-4-acetyl benzene in 25ml of THF was added dropwise and the reaction mixture was stirred for 1hr at −78° C., then 4.0 ml (19.42 mmole) of diethylchlorophosphate wereadded by syringe. After 15 min cooling bath was removed and the reactionmixture was allowed to heat up to RT and stirred for 3 hrs. The resultedmixture was cooled again to −78° C. and 29 ml of 2.0 M LDA solution wereadded dropwise. At the end of the addition the reaction mixture wasallowed to warm up to RT and stirred for additional 3 hrs. After thatperiod of time it was cooled again to −20° C., 9.0 ml (70.91 mmole) oftrimethylsilyl chloride were injected and the stirring was continued for2 hrs at RT. The reaction mixture was poured into 200 ml of ice/sodiumbicarbonate saturated aqueous solution and 300 ml of ether were added toextract organic compounds. The aqueous phase was separated and extractedagain with 2×100 ml of ether. The ether fractions were combined, driedover sodium sulfate and evaporated. The resulted liquid residue waspurified by silica gel chromatography (100% n-hexane as eluent). 4.1 gr(54% yield) were obtained.

[0279] Synthesis of Product (P 3). To a solution of 1.14 gm of Compound#3 (as described above) and 1.60 gm of P 2 in 100 mL of diethylaminewere added 0.23 gm of [1,1′-bis(diphenylphosphino)ferrocene]palladium(II) chloride and 0.1 gm of copper (I) iodide under Argon. The reactionmixture was stirred at 55° C. for 1 h and stirred at room temperatureovernight. After removing the solvent, the residue was dissolved indichloromethane for column separation. Silica gel (120 gm) was packedwith 20% ethyl acetate/CH₂Cl₂. The sample solution was loaded and thecolumn was eluted with 20-50% ethyl acetate/CH₂Cl₂. The fractions wereidentified by TLC (EtOAC: CH2C12=50:50) and pooled and concentrated togive 1.70 gm (84.0%) of TMS-derivative of P 3.

[0280] To a solution of 0.74 gm of TMS-derivative of P 3 in 70 mL ofdichloromethane at 0° C. was added 2.2 mL of 1.0 M (nBu)₄NF THFsolution. After stirring for 30 min, the solution was washed once withwater and dried over sodium sulfate. The solvent was removed, theresidue was used for column separation. Silica gel (20 gm) was packedwith 20% ethyl acetate/CH₂Cl₂, the column was eluted with 20-40% ethylacetate/CH₂Cl₂. The fractions containing the fluorescent compound werecombined and concentrated to dryness to afford 0.5 gm (81.3%) of thepure P 3.

[0281] Synthesis of P 4: To a solution of 0.5 gm of P 3 and 0.63 gm of P2 in 50 mL of dry DMF and 10 mL of TEA were added 100 mgm of[1,1′-bis(diphenylphosphino)ferrocene]palladium (II) chloride and 50 mgmof copper (I) iodide under Argon. The reaction mixture was stirred at55° C. for 1 h and stirred at 35° C. overnight. The solvents wereremoved in vacuo and the residue was dissolved in 10 mL of CH₂Cl₂ forcolumn separation. Silica gel (100 gm) was packed with 20% ethylacetate/CH₂Cl₂, after loading the sample, the column was eluted with20-40% ethyl acetate/CH₂Cl₂. The fractions were identified by TLC(EtOAC: CH₂Cl₂=50:50) and pooled and concentrated to give 0.47 gm(61.3%) of TMS-derivative of P 4.

[0282] To a solution of 0.47 gm of TMS-derivative of P 4 in 70 mL ofdichloromethane at 0° C. was added 1.0 mL of 1.0 M (nBu)₄NF THFsolution. After stirring for 30 min, the solution was washed once withwater and dried over sodium sulfate. The solvent was removed, theresidue was used for column separation. Silica gel (20 gm) was packedwith 20% ethyl acetate/CH₂Cl₂, the column was eluted with 20-40% ethylacetate/CH₂Cl₂. The fractions containing the fluorescent compound werecombined and concentrated to dryness to afford 0.32 gm (78.7%) of thepure P 4.

Example 4 Synthesis of a Nucleoside with a Metallocene Second ElectronTransfer Moiety Attached via a Ribose

[0283] Synthesis of 5′-O-DMT-2′-deoxy-2′-(ferrocenecarbonyl)aminoUridine (UAF): To a solution of 2.5 gm (10.9 mmol) of ferrocenemonocarboxylic acid in 350 mL of dichloromethane were added 2.25 gm(10.9 mmol) of DCC and 1.27 gm (10.9 mmol) of N-hydroxysuccinimide. Thereaction mixture was stirred for 3 h and the precipitate was formed. Theprecipitate was filtered off and washed once with dichloromethane. Thecombined filtrate was added into 4.5 gm (8.25 mmol) of2′-deoxy-2′-amino-5′-O-DMT uridine, followed by adding 2 mL oftriethylamine. The reaction mixture was stirred at room temperature for8 days. After eremoving the solvent, the residure was dissolved indichloromethane for separation. Silica gel (120 gm) was packed with 1%TEA/2% CH₃OH/CH₂Cl₂. After loading the sample solution, the column waseluted with 2-7% CH₃OH/1% TEA/CH₂Cl₂. The fraction was identified byTLC(CH₃OH: CH₂Cl₂=1:9) and pooled and concentrated to dryness to afford1.3 gm(22.0%) of the title compound.

[0284] Synthesis of UAF Phosphoramidite:

[0285] Preparation of Diisopropylaminochloro(β-cyano)ethoxyphosphine: Toa solution of 0.54 mL(4.0 mmol) of dichloro(β-cyano)ethoxyphosphine in40 mL of dichloromethane cooled in an ice-water bath was added 10 mL ofdiisopropylethylamine, followed by adding 0.64 mL (4.0 mmol) ofdiisopropylamine under Argon. The reaction mixture was warmed up to roomtemperature and stirred for 2 h. After adding 0.1 gm of DMAP into thesolution, the reaction mixture is ready for the next step reaction.

[0286] Preparation of UAF phosphoramidite: To a solution of 1.30 gm(1.72 mmol) of 5′-O-DMT-5-ferrocenylacetylenyl-2′-deoxy uridine in 40 mLof dichloromethane cooled in an ice-water bath was added 10 mL ofdiisopropylethylamine. The prepared phosphine solution was transferredinto the nucleoside solution through a syringe. The reaction mixture waswarmed up to room temperature and stirred overnight. The solution wasdiluted by adding 100 mL of dichloromethane and washed once with 200 mLod 5% aqueous NaHCO₃ solution, and once with the brine (200 mL) anddried over Na₂SO₄ and concentrated to dryness. Silica gel(47 gm) waspacked with 2% TEA/1% CH₃OH/CH₂Cl₂. The residue was dissolved in 10 mLof dichloromethane and loaded. The column was eluted with 150 mL of 1%TEA/1% CH₃OH/CH₂Cl₂ and 250 mL of 1% TEA/2% CH₃OH/CH₂Cl₂. The fractionswere pooled and concentrated to give 0.5 gm (30.3%) of the titlecompound.

[0287] Nucleotides containing conductive oligomers and second electrontransfer moieties were incorporated into nucleic acids using standardnucleic acid synthesis techniques; see “Oligonucleotides and Analogs, APractical Approach”, Ed. By F. Eckstein, Oxford University Press, 1991,hereby incorporated by reference.

Example 5 Synthesis of a Nucleoside with a Metallocene Second ElectronTransfer Moiety Attached via the Base

[0288] Synthesis of 5′-O-DMT-5-ferrocenylacetylenyl-2′-deoxy uridine(UBF): In a flask were added 4.8 gm(13.6 mmol) of 5-iodo-2′-deoxyuridine, 400 mg of bis(triphenylphosphine)palladium (II) chloride, 100mg of cuprous iodide, 95 mL of DMF and 10 mL of TEA. The solution wasdegassed by Argon and the flask was sealed. The reaction mixture wasstirred at 50° C. overnight. After removing solvents in vacuo, theresidue was dissolved in 140 mL of dry pyridine, followed by adding 0.2gm of DMAP and 5.0 gm (14.8 mmol) of DMT-Cl. The reaction mixture wasstirred at RT overnight. After removing the solvent, the residue wasdissolved in 300 mL of dichlromethane and washed twice with 5% aqueousNaHCO₃ (2×200 mL), twice with the brine (2×200 mL) and dried over sodiumsulfate. The solvent was removed and the residue was coevaporated twicewith toluene and dissolved in 15 mL of dichloromethane for columnseparation. Silica gel (264 gm) was packed 0.5% TEA/CH₂Cl₂. Afterloading the crude product solution, the column was eluted with 300 mL of1% TEA/2% CH₃OH/CH₂Cl₂, 400 mL of 1% TEA/5% CH₃OH/CH₂Cl₂, and 1.2 L of1% TEA/7% CH₃OH/CH₂Cl₂. The fractions were identified by TLC(CH₃OH:CH₂Cl₂=10:90) and pooled and concentrated to dryness to give 7.16 gm(71.3%) of the title compound.

[0289] Synthesis of UBF Phosphoramidite:

[0290] Preparation of Diisopropylaminochloro(β-cyano)ethoxyphosphine: Toa solution of 1.9 mL(13.8 mmol) of dichloro(β-cyano)ethoxyphosphine in40 mL of dichloromethane cooled in an ice-water bath was added 10 mL ofdiisopropylethylamine, followed by adding 2.3 mL (13.8 mmol) ofdiisopropylamine under Argon. The reactionb mixture was warmed up toroom temperature and stirred for 2 h. After adding 0.1 gm of DMAP intothe solution, the reaction mixture is ready for next step reaction.

[0291] Preparation of UBF phosphoramidite: To a solution of 3.42 gm(4.63 mmol) of 5′-O-DMT-5-ferrocenylacetylenyl-2′-deoxy uridine in 40 mLof dichloromethane cooled in an ice-water bath was added 10 mL ofdiisopropylethylamine. The prepared phosphine solution was transferredinto the nucleoside solution through a syringe. The reaction mixture waswarmed up to room temperature and stirred overnight. The solution wasdiluted by adding 150 mL of dichloromethane and washed once with 200 mLof 5% aqueous NaHCO₃ solution, and once with the brine (200 mL) anddried over Na₂SO₄ and concentrated to dryness. Silica gel(92 gm) waspacked with 2% TEA/i % CH₃OH/CH₂Cl₂. The residue was dissolved in 10 mLof dichloromethane and loaded. The column was eluted with 500 mL of 1%TEA/2% CH₃OH/CH₂Cl₂. The fractions were pooled and concentrated to give3.0 gm (69.0%) of the title compound.

[0292] Nucleotides containing conductive oligomers and second electrontransfer moieties were incorporated into nucleic acids using standardnucleic acid synthesis techniques; see “Oligonucleotides and Analogs, APractical Approach”, Ed. By F. Eckstein, Oxford University Press, 1991,hereby incorporated by reference.

Example 5 Synthesis of an Electrode Containing Nucleic Acids ContainingConductive Oligomers with a Monolayer of (CH₂)₁₆

[0293] Using the above techniques, and standard nucleic acid synthesis,the uridine with the phenyl-acetylene conductive polymer of Example 1was incorporated at the 3′ position to form the following nucleic acid:ACCATGGACTCAGCU-conductive polymer of Example 1 (hereinafter “wire-1”).

[0294] HS-(CH2)16-OH (herein “insulator-2”) was made as follows.

[0295] 16-Bromohexadecanoic acid. 16-Bromohexadecanoic acid was preparedby refluxing for 48 hrs 5.0 gr (18.35 mmole) of 16-hydroxyhexadecanoicacid in 24 ml of 1:1 v/v mixture of HBr (48% aqueous solution) andglacial acetic acid. Upon cooling, crude product was solidified insidethe reaction vessel. It was filtered out and washed with 3×100 ml ofcold water. Material was purified by recrystalization from n-hexane,filtered out and dried on high vacuum. 6.1 gr (99% yield) of the desiredproduct were obtained.

[0296] 16-Mercaptohexadecanoic acid. Under inert atmosphere 2.0 gr ofsodium metal suspension (40% in mineral oil) were slowly added to 100 mlof dry methanol at 0° C. At the end of the addition reaction mixture wasstirred for 10 min at RT and 1.75 ml (21.58 mmole) of thiolacetic acidwere added. After additional 10 min of stirring, 30 ml degassedmethanolic solution of 6.1 gr (18.19 mmole) of 16-bromohexadecanoic acidwere added. The resulted mixture was refluxed for 15 hrs, after which,allowed to cool to RT and 50 ml of degassed 1.0 M NaOH aqueous solutionwere injected. Additional refluxing for 3 hrs required for reactioncompletion. Resulted reaction mixture was cooled with ice bath andpoured, with stirring, into a vessel containing 200 ml of ice water.This mixture was titrated to pH=7 by 1.0 M HCl and extracted with 300 mlof ether. The organic layer was separated, washed with 3×150 ml ofwater, 150 ml of saturated NaCl aqueous solution and dried over sodiumsulfate. After removal of ether material was purified byrecrystalization from n-hexane, filtering out and drying over highvacuum. 5.1 gr (97% yield) of the desired product were obtained.

[0297] 16-Bromohexadecan-1-ol. Under inert atmosphere 10 ml of BH₃.THFcomplex (1.0 M THF solution) were added to 30 ml THF solution of 2.15 gr(6.41 mmole) of 16-bromohexadecanoic acid at −20° C. Reaction mixturewas stirred at this temperature for 2 hrs and then additional 1 hr atRT. After that time the resulted mixture was poured, with stirring, intoa vessel containing 200 ml of ice/saturated sodium bicarbonate aqueoussolution. Organic compounds were extracted with 3×200 ml of ether. Theether fractions were combined and dried over sodium sulfate. Afterremoval of ether material was dissolved in minimum amount ofdicloromethane and purified by silica gel chromatography (100%dicloromethane as eluent). 1.92 gr (93% yield) of the desired productwere obtained.

[0298] 16-Mercaptohexadecan-1-ol. Under inert atmosphere 365 mg ofsodium metal suspension (40% in mineral oil) were added dropwise to 20ml of dry methanol at 0° C. After completion of addition the reactionmixture was stirred for 10 min at RT followed by addition of 0.45 ml(6.30 mmole) of thiolacetic acid. After additional 10 min of stirring 3ml degassed methanolic solution of 1.0 gr (3.11 mmole) of16-bromohexadecan-1-ol were added. The resulted mixture was refluxed for15 hrs, allowed to cool to RT and 20 ml of degassed 1.0 M NaOH aqueoussolution were injected. The reaction completion required additional 3 hrof reflux. Resulted reaction mixture was cooled with ice bath andpoured, with stirring, into a vessel containing 200 ml of ice water.This mixture was titrated to pH=7 by 1.0 M HCl and extracted with 300 mlof ether. The organic layer was separated, washed with 3×150 ml ofwater, 150 ml of saturated NaCl aqueous solution and dried over sodiumsulfate. After ether removal material was dissolved in minimum amount ofdicloromethane and purified by silica gel chromatography (100%dicloromethane as eluent). 600 mg (70% yield) of the desired productwere obtained.

[0299] A clean gold covered microscope slide was incubated in a solutioncontaining 100 micromolar HS—(CH₂)₁₆—COOH in ethanol at room temperaturefor 4 hours. The electrode was then rinsed throughly with ethanol anddried. 20-30 microliters of wire-1 solution (1 micromolar in 1×SSCbuffer at pH 7.5) was applied to the electrode in a round droplet. Theelectrode was incubated at room temperature for 4 hours in a moistchamber to minimize evaporation. The wire-1 solution was then removedfrom the electrode and the electrode was immersed in 1×SSC bufferfollowed by 4 rinses with 1×SSC. The electrode was then stored at roomtemperature for up to 2 days in 1×SSC.

[0300] Hybridization efficiency was determined using ³²P complementaryand noncomplementary 15 mers corresponding to the wire-1 sequence. Theelectrodes were incubated with 50 microliters of each of the labellednon-complementary (herein “A5”) or complementary (herein “S5”) targetsequences applied over the entire electrode in 1×SSC as depicted inTable 1. The electrodes were then incubated for 1-2 hours at roomtemperature in a moist chamber, and rinsed as described above. Theamount of radiolabelled DNA was measured for each electrode in ascintllation counter, and the electrodes were dried and exposed to X-rayfilm for 4 hours. TABLE 1 ³²P counts total ³²P counts hybridized tohybridized with: added surface A5, 20% specific activity, DNA 46,446 152concentration 1 nM, 1 hour incubation S5, 30% specific activity, DNA39,166 10,484 concentration 1 nM, 1 hour (27% incubation hybridized) A5,14% specific activity, DNA 182,020 172 concentration 5 nM, 2 hourincubation S5, 20% specific activity, DNA 96,284 60,908 concentration 5nM, 2 hour (63% incubation hybridized)

Example XX Synthesis of Compositions Containing Ferrocene Linked to anElectrode

[0301] It has been shown in the literature that cyclic voltametry can beused to determine the electron transfer rate of surface bound molecules.Surface bound molecules should show perfectly symetric oxidation andreduction peaks if the scan speed of the voltammagram is sufficientlyslow. As the scan rate is increased, these peaks are split apart due tothe kinetics of electron transfer through the molecules. At a given scanspeed, a poorly conducting molecule should exhibit greater splittingthan a good conductor. As the speed is increased, the poor conductorwill be split even more.

[0302] Accordingly, to test the conductivity of the conductive polymeras compared to a traditional insulator, two molecules were tested. Thesynthesis of ferrocene attached via a conductive oligomer to anelectrode (herein “wire-2”) was made as follows, as depicted in FIG. 7.

[0303] Synthesis of compound #11 was as follows. 2.33 gr (5.68 nmole) ofcompound #10 (made as described in Hsung et al., Organometallics14:4808-4815 (1995), incorporated by reference), 90 mg (0.47 mmole) ofCuI and 80 mg (0.11 mmole) of PdCl₂(PPh₃)₂ were dissolved in 100 ml ofpyrrolidine under inert atmosphere and heated for 20 hrs at 50° C. Allvolatile components were removed on high vacuum and resulted cruderesidue was dissolved in minimum amount of dichloromethane. The desiredcompound was purified by silica gel chromatography (50% ethylacetate+50% dichloromethane as eluent). 3.2 gr (90% yield) of the pureproduct were obtained.

[0304] Compound #12. To 200 mg (0.32 mmole) of suspension of MG#1 in 200ml of acetone (sonication was applied in order to get better results) 3ml of MeI were added and the reaction mixture was stirred for 20 hrs atRT. After that time volume of the resulted solution was reduced byrotovap evaporation to 50 ml and then 400 ml of n-hexane were added.Formed precipitate was filtered out, washed with 3×200 ml of n-hexaneand dried on high vacuum. Quantitative yield of the desired compound wasobtained.

[0305] Compound #13. To 100 mg (0.13 mmole) of suspension of MG#2 in 200ml of acetone (sonication was applied in order to get better results) 10ml of triethyl amine were added and the reaction mixture was stirred for20 hrs at RT. After that time volume of the resulted solution wasreduced by rotovap evaporation to 50 ml and then 400 ml of n-hexane wereadded. Formed precipitate was filtered out, washed with 3×200 ml ofn-hexane and dried on high vacuum. The desired compound was extractedfrom this precipitate with 3×50 ml of THF.

[0306] Evaporation of the THF fractions gave 35 mg (52%) of the compound#13. This was then added to a gold electrode as known in the art.

[0307] HS—(CH2)15NHCO-Fc (herein “insulator-1”) was made as described inWard et al., Anal. Chem. 66:3164-3172 (1994), hereby incorporated byreference (note: the FIG. 1 data has been shown to be incorrect,although the synthesis of the molecule is correct).

[0308] Monolayers of each were made as follows. Insulator: Gold coveredmicroscope slides were immersed in a mixture of insulator-1 andHS-(CH2)15—OH (insulator-2) in neat ethanol. Insulator-2 molecule isadded to the mixture to prevent the local concentration of ferrocene atany position from being too high, resulting in interactions between theferrocene molecules. The final solution was 0.1 mM insulator-1 and 0.9mM insulator-2. The mixture was sonicated and heated (60-80° C.) for1-10 hours. The electrodes were rinsed thoroughly with ethanol, waterand ethanol. The electrodes were immersed in a 1 mM thiol solution inneat ethanol and let stand at room temperature for 2-60 hours. Theelectrodes were then rinsed again. This procedure resulted in 1-10%coverage of insulator-1 as compared to calculated values of close packedferrocene molecules on a surface. More or less coverage could easily beobtained by altering the mixture concentration and/or incubation times.

[0309] Wires: The same procedure was followed as above, except that thesecond step coating required between 10 and 60 hours, with approximately24 hours being preferable. This resulted in lower coverages, withbetween 0.1 and 3% occurring.

[0310] Cyclic voltametry was run at 3 scan speeds for each compound:IV/sec, 10 V/ec, and 50 V/sec. Even at 1 V/sec, significant splittingoccurs with insulator-1, with roughly 50 mV splitting occuring. Athigher speeds, the splitting increases. With wire-2, however, perfectlysymmetrical peaks are observed at the lower speeds, with only slightsplitting occurring at 50 V/sec.

[0311] It should be noted that despite a significant difference inelectron transfer rate, electron transfer does still occur even inpoorly conducting oligomers such as (CH₂)₁₅, traditionally called“insulators”. Thus the terms “conductive oligomer” and “insulator” aresomewhat relative.

Example XX Synthesis and Analysis of Nucleic Acid with Both a ConductiveOligomer and a Second Electron Transfer Moiety

[0312] The following nucleic acid composition was made using thetechniques above:

[0313] 5′-ACCATGGAC[UBF]CAGCU-conductive polymer (Structure 5 type, asoutlined above) herein “wire-3”, with UBF made as described above. Thus,the second electron transfer moiety, ferrocene, is on the sixth basefrom the conductive oligomer.

[0314] Mixed monolayers of wire-3 and insulator-2 were constructed usingthe techniques outlined above. The compositions were analyzed in 0.2 MNaClO₄ in water using cyclic voltametry (CV) and square wave voltametry(SW), in the absence (i.e. single stranded) and presence (i.e. doublestranded) of complementary target sequence.

[0315] The results of SW show the absence of a peak prior tohybridization, i.e. in the absence of double stranded nucleic acid. Inthe presence of the complementary target sequence, a peak at ˜240 mV,corresponding to ferrocene, was seen.

[0316] A mediator as described herein was also used. 6 mM ferricyanide(Fe(CN)₆) was added to the solution. Ferricyanide should produce a peakat 170 mV in a SW experiment. However, no peak at 170 mV was observed,but the peak at 240 mV was greatly enhanced as compared to the absenceof ferricyanide.

[0317] Alternatively, CV was done. No peaks were observed in the absenceof target sequence. Once again, the chip was incubated with perfectlycomplimentary nucleic acid in order to hybridize the surface nucleicacid. Again, the chip was scanned under the same conditions. Anincreased signal was observed. Finally, the chip was soaked in buffer at70° C. in order to melt the compliment off the surface. Previousexperiments with radioactive probes have shown that 15-mers hybridizedon a very similar surface melted at approximately 45° C. Repeating thescan after the heat treatment shows a reduced signal, as in the firstscan prior to hybridization.

We claim:
 1. A composition comprising: a) an electrode; b) at least onenucleoside; and c) a conductive oligomer covalently attached to bothsaid electrode and said nucleoside, wherein said conductive oligomer hasthe formula:

wherein Y is an aromatic group; n is an integer from 1 to 50; g iseither 1 or zero; e is an integer from zero to 10; and m is zero or 1;wherein when g is 1, B-D is a conjugated bond; and wherein when g iszero, e is 1 and D is preferably carbonyl, or a heteroatom moiety,wherein the heteroatom is selected from oxygen, sulfur, nitrogen orphosphorus.
 2. A composition comprising: a) an electrode; b) at leastone nucleoside; and c) a conductive oligomer covalently attached to bothsaid electrode and said nucleoside, wherein said conductive oligomer hasthe formula:

wherein n is an integer from 1 to 50; m is O or 1; C is carbon; J iscarbonyl or a heteroatom moeity, wherein the heteroatom is selected fromthe group consisting of nitrogen, silicon, phosphorus, sulfur; and G isa bond selected from alkane, alkene or acetylene.
 3. A compositionaccording to claim 1 or 2 wherein said nucleoside is part of a nucleicacid.
 4. A composition according to claim 3 further comprising aplurality of conductive oligomers each covalently attached to a nucleicacid.
 5. A composition according to claim 4 wherein said nucleic acidsare all the same.
 6. A composition according to claim 4 wherein at leastone of said nucleic acids is different.
 7. A composition according toclaim 1 or 2 wherein said covalent attachment of said conductiveoligomer to said nucleoside is to the ribose or phosphate of saidnucleoside.
 8. A composition according to claim 1 or 2 wherein saidcovalent attachment of said conductive oligomer to said nucleoside is tothe base of said nucleoside.
 9. A composition according to claim 1 or 2wherein said electrode further comprises at least one passavation agent.10. A composition according to claim 1 or 2 wherein said electrodefurther comprises a monolayer of passavation agents.
 11. A compositioncomprising: a) a first electron transfer moiety comprising an electrode;b) a nucleic acid; c) a second electron transfer moiety covalentlyattached to said nucleic acid; and d) a conductive oligomer covalentlyattached to both said electrode and said nucleic acid.
 12. A compositionaccording to claim 11 wherein said second electron transfer moietycomprises a transition metal complex.
 13. A composition according toclaim 11 wherein said second electron transfer moiety comprises anorganic electron transfer moiety.
 14. A composition according to claim11 wherein said covalent attachment of said second electron transfermoiety is to the ribose-phosphate backbone of said nucleic acid.
 15. Acomposition according to claim 11 wherein said covalent attachment ofsaid second electron transfer moiety is to a base of said nucleic acid.16. A composition according to claim 11 wherein said electrode furthercomprises at least one passavation agent.
 17. A composition according toclaim 11 wherein said electrode further comprises a monolayer ofpassavation agents.
 18. A composition according to claim 11 wherein saidconductive oligomer has the structure:

wherein Y is an aromatic group; n is an integer from 1 to 50; g iseither 1 or zero; e is an integer from zero to 10; and m is zero or 1;wherein when g is 1, B-D is a conjugated bond; and wherein when g iszero, e is 1 and D is preferably carbonyl, or a heteroatom moiety,wherein the heteroatom is selected from oxygen, sulfur, nitrogen orphosphorus.
 19. A composition according to claim 1 wherein saidconductive oligomer has the structure:

wherein n is an integer from 1 to 50; m is 0 or 1; C is carbon; J iscarbonyl or a heteroatom moeity, wherein the heteroatom is selected fromthe group consisting of nitrogen, silicon, phosphorus, sulfur; and G isa bond selected from alkane, alkene or acetylene.
 20. A method ofdetecting a target sequence in a nucleic acid sample comprising a)hybridizing a probe nucleic acid to said target sequence, if present, toform a hybridization complex, wherein said probe nucleic acid comprises:i) a conductive oligomer covalently attached to a first electrontransfer moiety comprising an electrode and to a single stranded nucleicacid capable of hybridizing to said target sequence, said singlestranded nucleic acid comprising a covalently attached second electrontransfer moiety, wherein said conductive oligomer has the formula:

 wherein Y is an aromatic group; n is an integer from 1 to 50; g iseither 1 or zero; e is an integer from zero to 10;and m is zero or 1;wherein when g is 1, B-D is a conjugated bond; and wherein when g iszero, e is 1 and D is preferably carbonyl, or a heteroatom moiety,wherein the heteroatom is selected from oxygen, sulfur, nitrogen orphosphorus; and b) detecting electron transfer between said electrodeand said second electron transfer moiety, if present, as an indicator ofthe present or absence of said target sequence.
 21. A method ofdetecting a target sequence in a nucleic acid sample comprising a)hybridizing a probe nucleic acid to said target sequence, if present, toform a hybridization complex, wherein said probe nucleic acid comprises:i) a conductive oligomer covalently attached to a first electrontransfer moiety comprising an electrode and to a single stranded nucleicacid capable of hybridizing to said target sequence, said singlestranded nucleic acid comprising a covalently attached second electrontransfer moiety, wherein said conductive oligomer has the formula:

 wherein n is an integer from 1 to 50; m is 0 or 1; C is carbon; J iscarbonyl or a heteroatom moeity, wherein the heteroatom is selected fromthe group consisting of nitrogen, silicon, phosphorus, sulfur; and G isa bond selected from alkane, alkene or acetylene; and b) detectingelectron transfer between said electrode and said second electrontransfer moiety, if present, as an indicator of the present or absenceof said target sequence.
 22. A method of detecting a target sequence ina nucleic acid wherein said target sequence comprises a first targetdomain and a second target domain, said method comprising: a)hybridizing a first probe nucleic acid to said first target domain, ifpresent, to form a hybridization complex, wherein said first probenucleic acid comprises: i) a conductive oligomer covalently attached toa first electron transfer moiety comprising an electrode and to a singlestranded nucleic acid capable of hybridizing to said target sequence,wherein said conductive oligomer has the formula:

 wherein Y is an aromatic group; n is an integer from 1 to 50; g iseither 1 or zero; e is an integer from zero to 10; and m is zero or 1;wherein when g is 1, B-D is a conjugated bond; and wherein when g iszero, e is 1 and D is preferably carbonyl, or a heteroatom moiety,wherein the heteroatom is selected from oxygen, sulfur, nitrogen orphosphorus; b) hybridizing a second single stranded nucleic acidcomprising a covalently attached electron transfer moiety to said secondtarget domain; and c) detecting electron transfer between said electrodeand said second electron transfer moiety, if present, as an indicator ofthe present or absence of said target sequence.
 23. A method ofdetecting a target sequence in a nucleic acid wherein said targetsequence comprises a first target domain and a second target domain,said method comprising: a) hybridizing a first probe nucleic acid tosaid first target domain, if present, to form a hybridization complex,wherein said first probe nucleic acid comprises: i) a conductiveoligomer covalently attached to a first electron transfer moietycomprising an electrode and to a single stranded nucleic acid capable ofhybridizing to said target sequence, wherein said conductive oligomerhas the formula:

 wherein n is an integer from 1 to 50; m is 0 or 1; C is carbon; J iscarbonyl or a heteroatom moeity, wherein the heteroatom is selected fromthe group consisting of nitrogen, silicon, phosphorus, sulfur; and G isa bond selected from alkane, alkene or acetylene; and b) hybridizing asecond single stranded nucleic acid comprising a covalently attachedelectron transfer moiety to said second target domain; and c) detectingelectron transfer between said electrode and said second electrontransfer moiety, if present, as an indicator of the present or absenceof said target sequence.
 24. A method for attaching a conductiveoligomer to a gold electrode comprising a) adding an ethyl pyridineprotecting group to a sulfur atom attached to a first subunit of saidconductive oligomer.
 25. A method according to claim 24, furthercomprising b) adding additional subunits to form said conductiveoligomer.
 26. A method according to claim 25, further comprising c)adding at least first nucleoside to said conductive oligomer.
 27. Amethod according to claim 26, further comprising d) adding additionalnucleosides to said first nucleoside to form a nucleic acid.
 28. Amethod according to claim 25 or 27, further comprising e) attaching saidconductive oligomer to said gold electrode.
 29. A conductive oligomerwith a ethyl-pyridine protected sulfur atom.
 30. A method of making acomposition according to claim 1, 2 or 11 comprising: a) providing aconductive oligomer covalently attached to a nucleoside; and b)attaching said conductive oligomer to said electrode.
 31. A method ofmaking a composition according to claim 1, 2 or 11 comprising: a)attaching a conductive oligomer to an electrode; and b) attaching atleast one nucleotide to said conductive oligomer.
 32. A compositioncomprising a conductive oligomer covalently attached to a nucleoside,wherein said conductive oligomer has the formula:

wherein Y is an aromatic group; n is an integer from 1 to 50; g iseither 1 or zero; e is an integer from zero to 10; and m is zero or 1;wherein when g is 1, B-D is a conjugated bond; and wherein when g iszero, e is 1 and D is preferably carbonyl, or a heteroatom moiety,wherein the heteroatom is selected from oxygen, sulfur, nitrogen orphosphorus.
 33. A composition comprising a conductive oligomercovalently attached to a nucleoside, wherein said conductive oligomerhas the formula:

wherein n is an integer from 1 to 50; m is 0 or 1; C is carbon; J iscarbonyl or a heteroatom moeity, wherein the heteroatom is selected fromthe group consisting of nitrogen, silicon, phosphorus, sulfur; and G isa bond selected from alkane, alkene or acetylene
 34. A compositioncomprising a conductive oligomer covalently attached to aphosphoramidite nucleoside, wherein said conductive oligomer has theformula:

wherein Y is an aromatic group; n is an integer from 1 to 50; g iseither 1 or zero; e is an integer from zero to 10; and m is zero or 1;wherein when g is 1, B-D is a conjugated bond; and wherein when g iszero, e is 1 and D is preferably carbonyl, or a heteroatom moiety,wherein the heteroatom is selected from oxygen, sulfur, nitrogen orphosphorus.
 35. A composition comprising a conductive oligomercovalently attached to a phosphoramidite nucleoside, wherein saidconductive oligomer has the formula:

wherein n is an integer from 1 to 50; m is 0 or 1; C is carbon; J iscarbonyl or a heteroatom moeity, wherein the heteroatom is selected fromthe group consisting of nitrogen, silicon, phosphorus, sulfur; and G isa bond selected from alkane, alkene or acetylene
 36. A compositioncomprising a conductive oligomer covalently attached to aCPG-nucleoside.
 37. A composition comprising a nucleoside covalentlylinked to a metallocene.
 38. A composition according to claim 37 whereinsaid metallocene is ferrocene or substituted ferrocene.
 39. Acomposition according to claim 37 wherein said metallocene is covalentlyattached to the base of said nucleoside.
 40. A composition comprising:a) an electrode; b) at least one metallocene; and c) a conductiveoligomer covalently attached to both said electrode and saidmetallocene, wherein said conductive oligomer has the formula:

wherein n is an integer from 1 to 50; m is 0 or 1; C is carbon; J iscarbonyl or a heteroatom moeity, wherein the heteroatom is selected fromthe group consisting of nitrogen, silicon, phosphorus, sulfur; and G isa bond selected from alkane, alkene or acetylene
 41. A compositioncomprising: a) an electrode; b) at least one metallocene; and c) aconductive oligomer covalently attached to both said electrode and saidmetallocene, wherein said conductive oligomer has the formula:

wherein Y is an aromatic group; n is an integer from 1 to 50; g iseither 1 or zero; e is an integer from zero to 10; and m is zero or 1;wherein when g is 1, B-D is a conjugated bond; and wherein when g iszero, e is 1 and D is preferably carbonyl, or a heteroatom moiety,wherein the heteroatom is selected from oxygen, sulfur, nitrogen orphosphorus.